Blood brain barrier device

ABSTRACT

Bone cages are disclosed including devices for biocompatible implantation. The structures of bone are useful for providing living cells and tissues as well as biologically active molecules to subjects.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is related to and claims the benefit of theearliest available effective filing date(s) from the following listedapplication(s) (the “Related Applications”) (e.g., claims earliestavailable priority dates for other than provisional patent applicationsor claims benefits under 35 USC §119(e) for provisional patentapplications, for any and all parent, grandparent, great-grandparent,etc. applications of the Related Application(s)).

RELATED APPLICATIONS

The present application constitutes a divisional application ofcurrently co-pending U.S. patent application Ser. No. 11/389,268,entitled BLOOD BRAIN BARRIER DEVICE, naming Ed Harlow; Roderick A. Hyde;Edward K.Y. Jung; Robert Langer; Eric C. Leuthardt and Lowell L. Wood,Jr. as inventors, and filed 24 Mar. 2006.

For purposes of the USPTO extra-statutory requirements, the presentapplication constitutes a continuation-in-part of U.S. patentapplication Ser. No. 11/304,486, entitled BONE DELIVERY DEVICE, namingEd Harlow; Roderick A. Hyde; Edward K. Y. Jung; Robert Langer; Eric C.Leuthardt and Lowell L. Wood, Jr. as inventors, filed 14 Dec. 2005,which is currently co-pending, or is an application of which a currentlyco-pending application is entitled to the benefit of the filing date.

For purposes of the USPTO extra-statutory requirements, the presentapplication constitutes a continuation-in-part of U.S. patentapplication Ser. No. 11/304,492, entitled BONE CELL DELIVER DEVICE,naming Ed Harlow; Roderick A. Hyde; Edward K. Y. Jung; Robert Langer;Eric C. Leuthardt and Lowell L. Wood, Jr. as inventors, filed 14 Dec.2005 now U.S. Pat. No. 7,855,062, which is currently co-pending, or isan application of which a currently co-pending application is entitledto the benefit of the filing date. For purposes of the USPTOextra-statutory requirements, the present application constitutes acontinuation-in-part of U.S. patent application Ser. No. 11/304,499,entitled BONE SEMI-PERMEABLE DEVICE, naming Ed Harlow; Roderick A. Hyde;Edward K. Y. Jung; Robert Langer; Eric C. Leuthardt and Lowell L. Wood,Jr. as inventors, filed 14 Dec. 2005, which is currently co-pending, oris an application of which a currently co-pending application isentitled to the benefit of the filing date.

The United States Patent Office (USPTO) has published a notice to theeffect that the USPTO's computer programs require that patent applicantsreference both a serial number and indicate whether an application is acontinuation or continuation-in-part. Stephen G. Kunin, Benefit ofPrior-Filed Application, USPTO Official Gazette Mar. 18, 2003, availableat http://www.uspto.gov/web/offices/com/sol/og/2003/week11/patbene.htm.The present applicant entity has provided above a specific reference tothe application(s) from which priority is being claimed as recited bystatute. Applicant entity understands that the statute is unambiguous inits specific reference language and does not require either a serialnumber or any characterization, such as “continuation” or“continuation-in-part,” for claiming priority to U.S. patentapplications. Notwithstanding the foregoing, applicant entityunderstands that the USPTO's computer programs have certain data entryrequirements, and hence applicant entity is designating the presentapplication as a continuation-in-part of its parent applications as setforth above, but expressly points out that such designations are not tobe construed in any way as any type of commentary and/or admission as towhether or not the present application contains any new matter inaddition to the matter of its parent application(s).

All subject matter of the Related Applications and of any and allparent, grandparent, great-grandparent, etc. applications of the RelatedApplications is incorporated herein by reference to the extent suchsubject matter is not inconsistent herewith.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are schematic representations of an illustrative bonecage.

FIG. 1A shows an exterior view, including an optional semi-permeablemembrane on one part. FIG. 1B shows a cross-sectional view.

FIGS. 2A, 2B, and 2C are schematic representations of a bone cage thatpartially surrounds the internal cavity. In FIG. 2A, the bone cage isshown with a buckeyball shape. In FIG. 2B, the bone cage is shown with abarrel-like lattice work configuration. In FIG. 2C, the bone cage isshown with large cut-outs in the walls.

FIGS. 3A, 3B, and 3C show bone cages with closable openings. In FIG. 3A,the opening is closed with a bone plug. In FIG. 3B, the opening is shownclosed using an overlapping petri dish type of closure. In FIG. 3C, theopening is shown closed by attaching two egg shell-like halves.

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, and 4G are tables describing diseases anddisorders that may be prevented, treated and/or ameliorated using one ormore bone cages. FIG. 4A is a table describing disorders of amino acidmetabolism. FIG. 4B is a table describing disorders of organic acidmetabolism. FIG. 4C is a table describing disorders of fatty acidmetabolism. FIG. 4D is a table describing disorders of purine andpyrimidine metabolism. FIG. 4E is a table describing lysosomal storagedisorders. FIG. 4F is a table describing disorders of urea formation.FIG. 4G is a table describing disorders of peroxisomal metabolism.

FIGS. 5A and 5B are schematic representations of an illustrative bloodbrain barrier device. FIG. 5A shows a cross-sectional view with theendothelial cell ablumenal surface oriented toward the exterior of thedevice. FIG. 5B shows a cross-sectional view with the endothelial cellablumenal surface oriented toward the internal cavity.

FIGS. 6A and 6B are schematic representations of an illustrative bloodcerebrospinal fluid device. FIG. 6A shows a cross-sectional view withthe epithelial cell apical surface oriented toward the exterior of thedevice. FIG. 6B shows a cross-sectional view with the epithelial cellapical surface oriented toward the internal cavity.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented here.

This disclosure is drawn, inter alia, to devices and methods fordelivering one or more biologically active molecules and/or one or moreliving cells or tissues to a subject.

In one aspect, the disclosure is drawn to a device comprising a bonecage designed to, configured to, and/or structured to at least partiallyor completely surround one or more biologically active molecules and/orone or more living cells or tissues. In some embodiments, the device isa structure comprised of bone. In some embodiments, the device isimplantable and/or biocompatible.

As used herein, the term “implantable” means able to be placed within asubject. The bone cage may be implanted by any method known in the artincluding, but not limited to, surgery, injection, suppository, andinhalation. The bone cage may be placed, for example, subcutaneously,intra-muscularly, intra-peritoneally, intra-venously, intra-arteriolar,in capillary beds, subdermally, intradermally, orally, rectally, ornasally. The bone cage may be implanted during a surgical procedure, ormay be injected using, for example, a hollow bore needle, such as thoseused for biopsies. Alternatively, injection may be by a gun, such asthose used for anesthetic darts. The bone cage can be implanted in anylocation in a subject appropriate for the desired treatment, suchlocations are well-known to health care workers including, but notlimited to, physicians and nurses, as well as veterinary, animalhusbandry, fish, game, zoo, bird, reptile, and exotic animal officials.

In some embodiments, the bone cage is implanted in well-vascularizedsoft tissue, including, but not limited to, liver, kidney, muscle, lung,cadiac and/or brain tissue. In other embodiments, the bone cage isimplanted in less well-vascularized tissue including, but not limitedto, joints, cartilage, and fat. In some embodiments, the bone cage isimplanted in bone or behind the blood brain barrier. In yet otherembodiments, the bone cage is implanted in the bladder, uterus, orvagina.

As used herein, the term “biocompatible” means a material the bodygenerally accepts without a significant immune response/rejection orexcessive fibrosis. In some embodiments, some immune response and/orfibrosis is desired. In other embodiments, vascularization is desired.In other embodiments, vascularization is not desired.

In some embodiments, the bone cage is implanted in a subject selectedfrom the group consisting of mammal, reptile, bird, amphibian, and fish.In some embodiments, the subject is selected from the group consistingof domesticated, wild, research, zoo, sports, pet, primate, marine, andfarm animals. In some embodiments, the animal is a mammal. In someembodiments, the mammal is a human. In other embodiments, the primate isa human. Animals further include, but are not limited to, bovine,porcine, swine, ovine, murine, canine, avian, feline, equine, or rodentanimals. Domesticated and/or farm animals include, but are not limitedto, chickens, horses, cattle, pigs, sheep, donkeys, mules, rabbits,goats, ducks, geese, chickens, and turkeys. Wild animals include, butare not limited to, non-human primates, bear, deer, elk, raccoons,squirrels, wolves, coyotes, opossums, foxes, skunks, and cougars.Research animals include, but are not limited to, rats, mice, hamsters,guinea pigs, rabbits, pigs, dogs, cats and non-human primates. Petsinclude, but are not limited to, dogs, cats, gerbils, hamsters, guineapigs and rabbits. Reptiles include, but are not limited to, snakes,lizards, alligators, crocodiles, iguanas, and turtles. Avian animalsinclude, but are not limited to, chickens, ducks, geese, owls, seagulls, eagles, hawks, and falcons. Fish include, but are not limited to,farm-raised, wild, pelagic, coastal, sport, commercial, fresh water,salt water, and tropical. Marine animals include, but are not limitedto, whales, sharks, seals, sea lions, walruses, penguins, dolphins, andfish.

As used herein, the term “cage” or “structure” means a rigid,semi-rigid, or otherwise structurally supportive structure with at leastone external wall, and at least one internal cavity within which, forexample, a semi-permeable membrane and/or one or more living cells ortissues and/or one or more biologically active molecules can be placed.In some embodiments, the one or more living cells or tissues and/or oneor more biologically active molecules do not include bone tissue. Theexternal wall can be any shape, including but not limited to, spherical,oval, rectangular, square, trapezoidal or modified versions of theseshapes. The internal cavity can also be any shape, including but notlimited to, spherical, oval, rectangular, square, trapezoidal ormodified versions of these shapes. Moreover, the internal cavity mayinclude one or more portions that may be in fluid or similarcommunication or may be isolated.

In some embodiments, the external wall is approximately any dimension,preferably an integer μm from 1 to 1,000 including, but not limited to,2 μm, 3 μm, 4 μm, 5 μm, 8 μm, 10 μm, 12 μm, 15 μm, 20 μm, 25 μm, 50 μm,100 μm, 200 μm, 300 μm, 500 μm, 600 μm, 800 μm and 1,000 μm. In otherembodiments, the external wall is approximately 1 μm to 1,000 μm, 2 μmto 500 μm, 3 μm to 250 μm, 4 μm to 100 μm, 5 μm to 50 μm, 5 μm to 10 μm,2 μm to 20 μm, 1 μm to 50 μm, 5 μm to 25 μm, or 2 μm to 8 μm in width.In some embodiments, the width is not uniform throughout the structure.

In some embodiments, the diameter of the internal cavity isapproximately any integer μm from 1 to 1,000 including, but not limitedto, 2 μm, 3 μm, 4 μm, 5 μm, 8 μm, 10 μm, 12 μm, 15 μm, 20 μm, 25 μm, 50μm, 100 μm, 200 μm, 300 μm, 500 μm, 600 μm, 800 μm or 1,000 μm. In otherembodiments, the diameter is approximately 1 μm to 1,000 μm, 2 μm to 800μm, 5 μm to 750 μm, 10 μm to 500 μm, 20 μm to 250 μm, 10 μm to 100 μm, 5μm to 50 μm, 1 μm to 10 μm, 2 μm to 20 μm, 1 μm to 50 μm, 50 μm to 500μm, or 250 μm to 1,000 μm in width. In some embodiments, the internaldiameter is not uniform throughout the structure.

In some embodiments, the external wall is porous. As used herein, theterm “porosity” is defined as the percentage of void space in a solid(Adv. Colloid Interface Sci. (1998) 76-77:341-72). It is a morphologicalproperty independent of the material. Porosity may be created by, forexample, salt leaching, gas foaming, phase separation, freeze-drying,and sintering, depending on the material used to fabricate the bonescaffold.

In some embodiments, the porosity is approximately any integerpercentage from 1% to 99% including, but not limited to, 2%, 3%, 4%, 7%,10%, 12%, 15%, 20%, 35%, 50%, 60%, 75%, and/or 90%. In otherembodiments, the porosity is approximately 1% to 99%, 1% to 15%, 3% to12%, 5% to 10%, 40% to 95%, 50% to 90%, 60% to 75%, 3% to 90%, 10% to75%, 15% to 90%, and 25% to 90%. In some embodiments, the porosity isnot uniform throughout the bone. The porosity of trabecular bone is 50%to 90%, while that of cortical bone is 3% to 12% (Biomaterials (2005)26:5474-5491). In some embodiments, the pore size is approximately anyinteger nm from 1 to 10,000 including, but not limited to, 2 nm, 3 nm, 4nm, 5 nm, 8 nm, 10 nm, 12 nm, 15 nm, 20 nm, 25 nm, 50 nm, 100 nm, 200nm, 300 nm, 500 nm, 600 nm, 800 nm, 1,000 nm, 2,000 nm, 5,000 nm, or10,000 nm. In other embodiments, the pore size is approximately 1 nm to10,000 nm, 10 nm to 5,000 nm, 25 nm to 1,000 nm, 50 nm to 750 nm, 100 nmto 500 nm, 10 nm to 100 nm, 5 nm to 50 nm, 1 nm to 10 nm, 2 nm to 20 nm,500 nm to 5,000 nm, 1,000 nm to 10,000 nm, or 250 nm to 1,000 nm inwidth. In some embodiments, the pore size is not uniform throughout thestructure.

In some embodiments, the bone cage completely surrounds the one or morebiologically active molecules and/or one or more living cells ortissues. Illustrative examples of bone cages that completely surroundthe one or more biologically active molecules and/or one or more livingcells or tissues is shown in FIG. 1. In FIG. 1A, a rectangular cage 100is depicted, showing a bone structure 110 with pores 120 partiallysurrounded by a semi-permeable component. 130 optionally comprised ofcells 140. FIG. 1B shows a cross-section of the rectangular cage 100,showing the optional exterior semi-permeable component 130 optionallycomprised of cells 140, and the optional interior semi-permeablecomponent 130, as well as the bone structure 110 with pores 120, and theinternal cavity 150 with optional cells 140.

In other embodiments, the bone cage partially surrounds the one or morebiologically active molecules and/or one or more living cells ortissues. As used herein, the term “partially surrounds” describes amaterial or other structure, such as an external wall of the bone cagethat surrounds less than 100% of a material, structure, region, or otherobject or location. An example of a material that may be partiallysurrounded is the one or more biologically active molecules and/or oneor more living cells or tissues in the internal cavity. The term “lessthan 100%” includes any integer percentage from 1% to 99%. Illustrativeintegers include, 10%, 25%, 50%, 75%, and 95%.

Examples of bone cages with external walls that partially surround theinternal cavity include, but are not limited to, those where theexternal wall is a lattice, and/or where there are openings in the wallthat are larger than the pore size of the bone. Examples of lattice workexternal walls include, but are not limited to, those patterned afterbuckeyballs.

Examples of external walls with openings include, but are not limitedto, those with openings designed to facilitate the placement of thesemi-permeable membrane, and/or the one or more biologically activemolecules, and/or the one or more living cells or tissues, for example,within the internal cavity. In some embodiments, the width of the one ormore openings in the external wall is approximately any integer μm from1 to 1,000 including, but not limited to, 2 μm, 3 μm, 4 μm, 5 μm, 8 μm,10 μm, 12 μm, 15 μm, 20 μm, 25 μm, 50 μm, 100 μm, 200 μm, 300 μm, 500μm, 600 μm, 800 μm and 1,000 μm. In other embodiments, the width isapproximately 1 μm to 1,000 μm, 2 μm to 800 μm, 5 μm to 750 μm, 10 μm to500 μm, 20 μm to 250 μm, 10 μm to 100 μm, 5 μm to 50 μm, 1 μm to 10 μm,2 μm to 20 μm, 1 μm to 50 μm, 50 μm to 500 μm, or 250 μm to 1,000 μm inwidth, and the length is the width of the external wall as describedabove.

Illustrative examples of bone cages that partially surround the one ormore biologically active molecules and/or one or more living cells ortissues is shown in FIG. 2. FIG. 2A shows a buckeyball shaped cage 201in which the pentagonal and hexagonal shapes are comprised of bone 210.FIG. 2B shows a barrel-like shape 202, in which the vertical andhorizontal members 210 are comprised of bone with pores in between 220.FIG. 2C shows a rectangular structure 203, comprised of a bone structure210 containing large openings as pores 220.

In some embodiments where the external wall has one or more openings,the openings are closable. As used herein, the term “closable” meansthat an opening or other structural feature is configured to becompletely or partially filled, such that the opening can be made nolonger larger than the pore size of the bone. In some embodiments, theclosure has a width sufficiently greater than the width of the openingto allow attachment to the external wall completely surrounding theopening, and can be secured by any method known in the art. In otherembodiments, the closure spans the entire width of the opening, and/orthe entire length. In some embodiments, the plug or closure is comprisedof bone, including but not limited to, anorganic, artificial,demineralized, cultured in vitro, autologous, allogeneic or xenogeneicbone, or any combination thereof.

Illustrative embodiments of a bone cage with closable openings are shownin FIG. 3. FIG. 3A shows a rectangular cage 301 comprised of bone 310containing pores 320 containing an opening 360 that connects with theinternal cavity 350. The opening 360 is closable by the insertion of aplug 370 made of bone 310 of a size to approximately entirely fill theopening. FIG. 3B shows the two open halves 304 and 306 of a petridish-shaped cage 302 made of bone 310 containing pores 320 in which onehalf 304 has a uniformly slightly smaller diameter than the other half306 so that the sides of 306 overlap the sides of 304 on closure suchthat an internal cavity 350 remains. The two halves 304 and 306 areoptionally secured by sliding a partial internally protruding edge 385under a partial externally protruding edge 380. On closing, 304 and 306are positioned such that 380 and 385 can slide past each other. Once theinternally protruding edge 385 is past the externally protruding edge380, the halves 304 and 306 are twisted such that externally andinternally protruding edges 380 and 385 align. FIG. 3C shows the twoopen halves 305 and 307 of an egg shell-shaped structure 303 made ofbone 310 comprising pores 320, where the edges 390 and 395 of the twohalves 305 and 307, respectively, optionally mate to allow a screw-typeseal, forming an internal cavity 350.

As used herein, the term “bone” encompasses all types of bone known inthe art, including but not limited to, organic, anorganic,demineralized, freeze-dried, and artificial bone. The bone may becultured in vitro, and/or genetically engineered. The bone may be livingor dead. The bone may be autologous, allogeneic, or xenogeneic withrespect to a subject within whom or which the bone is implanted. In someembodiments, the bone may be a combination of one or more of the typesof bone described above.

As used herein, the term “organic bone” encompasses all kinds of boneobtained from donors including cortical, trabecular and cancellous. Thebone may be autologous (autografts), allogeneic (allografts) orxenogeneic (xenografts) with respect to a subject within whom or whichthe bone is implanted. An autograft is a graft from one part of anindividual to another part of the same individual. An allograft is agraft between genetically different individuals within one species. Axenograft is a graft between individuals of different species.

In illustrative embodiments, the bone cage is comprised of autologousbone excised from the iliac crest, skull, or fibula, for example.Autologous grafts do not typically have immune rejection issues.

In other illustrative embodiments, the bone cage is comprised ofallogeneic bone harvested from a cadaver from any location, for example,and is typically frozen prior to re-implantation to decreaseimmunogenicity. Following an allograft, donor cells generally do notsurvive in the recipient (The Merck Manual, Sec. 12, Ch. 149,Transplantation). Examples include, but are not limited to, Allogro,Orthroblast, Opteform and Grafton.

In yet other illustrative embodiments, xenogeneic bone is obtained fromanimals and is used for xenografts in man. For example, SurgiboneUnilab, which is prepared from bovine bone, has been used to augmentautografts for hip revision surgery (Acta Orthop. (2005) 76:544-9).Studies of the immunological mechanisms underlying the rejection of pigorgans injected into primates has resulted in the development of novellines of genetically engineered pigs that are more immunologicallycompatible with man (J. Nephrol. (2003) 16(suppl 7):S16-21), and usefulfor bone xenografts.

In other embodiments, the bone cage is comprised of anorganic bone.Anorganic bone or anorganic bone matrix is well known in the art for usebone repair (Clin. Plast. Surg. (1994) 21:437-44; J. Long Term Eff. Med.Implants (1998) 8:69-78). As used herein, the term “anorganic bone oranorganic bone matrix” includes autologous, allogeneic, or xenogeneicbone with respect to a subject within whom or which the bone isimplanted that has been deorganified. Illustrative examples include, butare not limited to, Bio-Oss^(R) (Geistlich, Wolhusen, Switzerland),which is composed of anorganic bovine bone (Arch Oral. Biol. (2005) July29 Epub ahead of print), and an anorganic bone matrix described inBiomaterials ((2005) 26:5648-57).

In yet other embodiments, the bone cage is comprised of demineralizedbone. Demineralized bone allograft is known in the art for bone repair(Cell Tissue Bank (2005) 6:3-12). As used herein, the term“demineralized bone” includes autologous, allogeneic, or xenogeneic bonewith respect to a subject within whom or which the bone is implantedthat has been demineralized. An illustrative example of the use ofdemineralized, freeze-dried bone together with anorganic bovine bone formaxillary sinus grafting is presented in Int. J. Oral Maxillofac.Implants ((2003) 18:556-60).

Once the organic, anorganic, freeze-dried and/or demineralized bone isobtained, the cage can be created in a variety of ways known in the art.In illustrative embodiments, the bone is machined using, for example,microtomes such as the Leica SP 2600 (or 1600) Saw Microtome (LeicaMicrosystems Nussloch GmbH, Postfach 1120, Heidelberger Strasse 17-19,D-69226 Nussloch, Germany) that can slice bone to a finished thicknessof approximately 20-30 μm. Lasers, such as the YAG laser rod, can beused to cut bone with a minimum width of approximately 10 μm for deeperbeam penetrations and less than 1 μm for thin coatings (Laserod Inc.1846-A West 169^(th) Street, Gardena, Calif. 90247-5252). Microtweezers, such as those from MEMS Precision Instruments(http://memspi.com), can be used to assemble the pieces as necessary.Methods for preparing 2-50 μm thick sections of undecalcified hardtissues are known in the art (Histochem Cell. Biol. (2000) 113:331-339).

An illustrative example of a bone cage that could be constructed usingthese techniques is shown in FIG. 2C. Since bone is a tubular structure,sections could be sliced perpendicular to the tubular Haversian systemsthat make up cortically dense bone to produce very thin bone rings.These rings could then be further sectioned into barrel staves to form abarrel-shaped construct, laid side by side to form a tube-shapedconstruct, or overlapped to make smaller portal structures. Furtherholes and smaller cutting could create joints to allow the variouscomponents to fit together and be assembled using micro tweezers.

An illustrative example of a method to make bone cages of FIG. 1 and/orFIG. 3A is described below. The bone cage is constructed by excising aportion of cortical bone approximately 3 mm by 1 mm from the iliac crestof a subject using a microsaw. This portion of bone is thenmicromachined to a desired size, for example 30 μm by 90 μm using amicrosaw. The shape is rectangular, or smoothed to an oblong, althoughother shapes may be implemented. The interior cavity of the bone cage ishollowed using a micromachining laser, leaving an approximately 5 μmthick bone wall. The bone wall is perforated with 1 to 2 μm holes usinga micromachining laser. A second piece of bone is micromachined andshaped to form a bone cap or plug.

In another embodiment, bone cages are constructed by excising a portionof bone, followed by micromachining to the desired size and/or shape.The orientation of the construct is selected to align the natural poresof the bone to form a natural internal cavity for the bone cage. Theinterior cavity of the bone cage can be further refined using focusedbeam machining to enlarge or re-shape the interior cavity of the bonecage. Additional pores can be added as described above, if the naturalporosity of the bone is not sufficient to allow the desired amountand/or type of nutrients and/or other materials to reach and/or elutefrom the internal cavity.

The methods for making a bone cage described above are illustrative andare not intended to be limiting. In addition, it should be anticipatedthat these and other methods could be used in combination as well asseparately.

In other embodiments, the bone cage is comprised of biocompatible and/orimplantable artificial bone substitutes containing metals, ceramicsand/or polymers. Artificial bone scaffolding is known in the art for usein bone repair (Int. J. Oral Maxillofac. Surg. (2004) 33:325-332; Int.J. Oral Maxillofac. Surg. (2004) 33:523-530). As used herein, the term“artificial bone” includes any bone substitute composites or scaffoldsknown in the art with a structural rigidity substantially equal to orgreater than that of cartilage, and with pores that allow at least fluidpassage. In some embodiments, the pores allow passage of macromolecules,but not cells. In other embodiments the pores allow passage of cells aswell as macromolecules. As used herein, the term “passage” may includediffusion, release, extrusion, and/or migration.

The mechanical properties of naturally occurring bone, includingstiffness and tensile strength, are provided by the bone tissue“scaffold” which contains significant amounts of non-living material,such as organic minerals as well various proteins of the extracellularmatrix.

A variety of bone substitutes are used in tissue engineering to createscaffolds (Synthetic Biodegradable Polymer Scaffolds (1997) Boston,Mass.: Birkhauser; J. Biomed. Mater. Res. (2001) 54:162-171; Int. J.Oral Maxillofac. Surg. (2004) 33:523-530). These include, but are notlimited to, synthetic organic materials such as clinically usednondegradable and biodegradable and bioresorbable polymers includingpolyglycolide, optically active and racemic polylactides, polydioxanone,and polycaprolactone, polymers under clinical invenstigation includingpolyorthoester, polyanhydrides, and polyhydroxyalkanoate, early stagepolymeric biomaterials including ploy(lactic acid-co-lysine), as well asbiodegradable polymer ceramic scaffolds (J. Mater. Sci. Mater. Med.(2005) 16:807-19; Biomaterials (1998) 19:1405-1412). Examples include,but are not limited to, Cortoss, OPLA, and Immix.

Synthetic inorganic molecules are also used in scaffolding, includinghydroxyapatite, calcium/phosphate composites, calcium sulfate, and glassceramics (Biotechnol. Bioeng. (2005); J. Artif. Organs (2005) 8:131-136;J. Biomed. Mater Res. A. (2005) 68:725-734; J. Long Term Eff. Med.Implants (1998) 8:69-78). Examples include, but are not limited to,Osteograf, Norian SRS, ProOsteon, and Osteoset.

Organic materials of natural origin including collagen, fibrin, andhyaluronic acid are also used, as are inorganic material of naturalorigin including, for example, coralline hydroxyapatite. A variety ofmetals have been used in artificial scaffolds for bone, includingsilicon, titanium and aluminum (J. Biomed. Mater. Res. A. (2004)70:206-218; J. Biomed. Mater. Res. (2001) 56:494-503; J. Biomed. Mater.Res. A. (2005) 72:288-295).

In addition to the methods for making bone cages discussed above, designand prototyping of scaffolds can be performed digitally (Biomaterials(2002) 23:4437-4447; Int. J. Prothodont. (2002) 15:129-132), and thematerial can be processed as sponge-like sheets, gels, or highly complexstructures with intricate pores and channels (Ann. NY Acad. Sci. (2002)961:83-95). A biocompatible three-dimensional internal architecturalstructure with a desired material surface topography, pore size, channeldirection and trabecular orientation can be fabricated (Biomaterials(2002) 23:4437-4447). Fabrication of scaffolding can be accomplishedusing conventional manual-based fabrication techniques (Frontiers inTissue Engineering (1998) New York, Elsevier Science 107-120; J. Biomed.Mater. Res. (2000) 51:376-382; J. Biomater. Sci. Polymer. E. (1995) 7;23-38), or computer-based solid free form fabrication technologies (Br.J. Plast. Surg. (2000) 53:200-204), for example.

In some embodiments, the bone cage is comprised of cells cultured invitro including, but not limited to, stem cells, fibroblasts,endothelial cells, osteoblasts and/or osteoclasts. In some embodiments,the non-stem cells are isolated from a subject. Bone cell populationsmay be derived from all bone surfaces by a variety of techniques knownin the art, including mechanical disruption, explantation, and enzymedigestion (Tissue Eng. (1995) 1:301-308). Methods to culture and/orpropagate osteoprogenitor cells and/or osteoblast-like cells in vitroare also well known in the art (Int. J. Oral Maxillofac. Surg. (2004)33:325-332). Culture conditions for manufacturing bone tissue including,but not limited to, temperature, culture medium, biochemical andmechanical stimuli, fluid flow and perfusion, are known in the art (Int.J. Oral Maxillofac. Surg. (2004) 33:523-530).

In other embodiments, the non-stem cells are differentiated from stemcell including, but not limited to, fetal, embryonic, cord blood,mesenchymal and/or hematopoeitic. In some embodiments, the numbers ofstem cells are increased in number in culture in vitro prior todifferentiation. Methods for isolation, culturing and transplantation ofstem cells are known in the art (Fetal Diagn. Ther. (2004) 19:2-8; BestPract. Res. Clin. Obstet. Gynaecol. (2004) 18:853-875).

In illustrative embodiments, the stem cells are mesenchymal stem cells.Mesenchymal stem cells are multipotent cells found in several, perhapsmost, adult tissues (Blood (2005) 105:1815-1822). Mesenchymal stem cellscan be reliably isolated and cultured in therapeutic quantities (Bone(1992) 13:81-88), and several methods to isolate mesenchymal stem cellsfrom, for example, bone marrow, adipose tissue, and muscle, based on thephysical and immunological characteristics are known in the art (Basic &Clinical Pharmacology & Toxicology (2004) 95:209-; Ann. Biomed. Eng.(2004) 32:136-147). Mesenchymal stem cells are able to differentiateinto various lineages including osteoblasts in vitro (Science(1999)284:143-147; J. Cell Sci. (2000) 113:1161-11.66; Int. J. OralMaxillofac. Surg. (2004) 33:325-332).

In some embodiments, the bone cage is comprised of cells cultured invitro on bone scaffolding. In some embodiments, the bone scaffolding isdegradable in vitro and/or in vitro. Porosity and pore size of thescaffold are known to play a role in bone formation, osteogenesis andosteoconduction in vitro and in vivo, and methods of measuring andcontrolling porosity and pore size in artificial scaffolds are known inthe art (Biomaterials (2005) 26:5474-5491).

In illustrative embodiments, stem cells and/or osteoblast progenitorcells are propagated on scaffolds of a variety of shapes including,those shown in FIG. 2. The cells are grown until fusion, or partiallygrown to result in a lattice shape. The bone cells cultured in vitroinclude autclogous, allogeneic, or xenogeneic cells, with respect to asubject within whom or which the bone cage is implanted. An illustrativemethod of making a bone cage, such as bone cages of FIG. 3B or 3C, usingmesenchymal stem cells is described below. An artificial scaffold of,for example, degrable polymer, is laid down in the desired openlattice-work shape of the two halves of the bone structure. Expandedmesenchymal stem cells (autologous, allogeneic, or xenogeneic) arecultured in the latticework shapes, in vitro, and encouraged todifferentiate into osteoblasts. Once the cells have populated thelattice structure, other optional components of the bone device areadded and the device implanted.

In some embodiments, the bone cage comprises living tissue. As usedherein, the term “living tissue” refers to the presence of living bonecells such as, but not limited to, osteoblasts, or osteoclasts withinthe bone scaffold. As used herein, the term “living tissue” includesliving bone cells in artificial bone scaffolding. The living tissue canbe autologous, allogeneic, or xenogeneic, with respect to a subjectwithin whom or which the bone cage is implanted.

In some embodiments, the bone cage comprises dead tissue. As usedherein, the term “dead tissue” refers to the absence of living bonecell, such as, but not limited to, osteoblasts, or osteoclasts withinthe bone scaffold. The dead tissue can be autologous, allogeneic, orxenogeneic, with respect to a subject within whom or which the bone cageis implanted.

In some embodiments, the bone cage is designed and/or treated to, atleast partially or completely, prevent restructuring. As used herein,the term “restructuring or restructured” as it relates to the bone cagemeans a change in the physical structure of the bone cage, including butnot limited to, bone size, shape, architecture and quality. Bonerestructuring includes, but is not limited to, bone resorption andosteoconduction (or bone deposition). In the case of a bone cage withartificial scaffolding, autologous, or non-autologous bone, bonerestructuring would include, but not be limited to, the influx andgrowth of the subject's bone cells in the artificial, autologous, ornon-autologous bone scaffold. Mechanisms of restructuring, treatments tomodify restructuring, and genes governing restructuring are known in theart (Nature (2005) 1:47-54).

Methods for detecting and measuring changes in bone are well-known inthe art. The change can result, for example, from global or discreteincreases or decreases in bone mass. Alternatively, the change canresult, for example, from global or discrete increases or decreases inthe relative ratios of cells, including but not limited to, the numberof osteoblasts as compared with the number of osteoclasts. The changecan also result, for example, from global or discrete increases ordecreases in bone pore size and/or porosity. As used herein, the terms“increase” and/or “decrease” in bone mass, relative ratio of cells, orpore size and/or porosity, for example, are measured as any integerpercent change from 1% to 99% as compared with the original bone mass,relative ratio of cells, or pore size and/or porosity, respectively,either globally or in a discrete location. Illustrative integers include10%, 25%, 50%, 75%, and 95%.

Bone restructuring, a combination of bone resorption by osteoclasts andbone deposition by osteoblasts, can be modified by methods known in theart. As used herein, the term “resorption” as it relates to the bonecage means a decrease in bone mass from either global or discretereductions in, for example, the extracellular matrix and/or cells. Boneresorption is mediated by osteoclasts, so treatments that inhibit theactivity of osteoclasts decrease bone resorption. Methods for detectingand measuring these changes are well-known in the art (Biomaterials(2005) 26:5474-5491).

In some embodiments, restructuring of the bone cage is partially orcompletely reduced or prevented. In other embodiments, the bone isdesigned and/or treated to be at least partially, or completely,restructured. Modifications of bone restructuring can result, forexample, from administration of compounds that influence bone resorptionand/or deposition, by the selection of the pore size and/or porosity ofthe bone, by the selection of the type of bone, by the selection of thelocation of implantation, as a result of inherent, induced, orgenetically modified immunogenicity, and as a result of other geneticmodification. In some embodiments, the bone is partially or completelyresorbable.

Compounds that influence bone restructuring through modifications inbone resorption and/or deposition can be applied before, during, orafter implantation of the bone cage at the discretion of the healthprofessional and depending on the timing and the extent of themodification of a subject's bone restructuring desired. Administrationof the compounds may be systemic or localized. Systemic and localadministration includes any method used in the art for pharmaceuticaladministration.

In illustrative embodiments, compounds can be administered locally bybeing applied to, or made part of, the bone either globally, or inlocalized areas, depending on whether complete or partial restructuringis desired. An illustrative example is the incorporation of the cellbinding peptide P-15 on anorganic bovine bone matrix (Biomaterials(2004) 25:4831-4836; J. Biomed. Mater. Res. A. (2005) 74:712-721;Biomaterials (2005) 26:5648-4657). Other examples include, but are notlimited to, addition of TGF-β, platelet-derived growth factor,fibroblast growth factor, and bone morphogenic proteins.

In other illustrative embodiments, compounds can be administered byincorporation in the bone cage as one of the one or more biologicallyactive molecules and/or living cells and/or tissues, as discussedherein.

In illustrative embodiments, bis-phosphonates, used systemically toprevent bone resorption (Osteoporos Int. (2002) 13:97-104), are appliedbefore, during, or after implantation of the bone cage to partially orcompletely modify bone restructuring (Curr. Osteoporos. Rep. (2003)1:45-52). Such therapies can also be administered locally by treatingthe bone cage, or by placing them inside the cage as one of the one ormore biologically active molecules and/or one or more living cells ortissues, to elute out over time. Alternatively, discrete portions of thebone cage could be coated to selectively prevent restructuring asdiscussed above.

In illustrative embodiments, one or more hormones including, but notlimited to, estrogen, growth hormone, calcitonin, vitamin D, and/orcalcium, which encourage bone growth, are administered before, during,or after implantation of the bone cage to partially or completely modifybone restructuring. In other embodiments, the bone cage is treatedglobally or discretely with a thin layer of one or more of thesehormones to encourage bone growth throughout or in discrete locations.

In yet other illustrative embodiments, anabolic therapies including, butnot limited to hormones such as parathyroidhormone (PTH-(1-84)),teriparatide (PTH-(1-34)), and/or excess glucocorticoid, that are knownto increase bone turnover and porosity are administered systemically(Osteoporosis Int. (2002) 13:97-104) to partially or completely modifyrestructuring. In other embodiments, these hormones are administeredlocally by treating the entire bone cage, or discrete portions of thebone cage, to allow selective restructuring. In yet other embodiments,these hormones are administered by placing them inside the cage as oneof the one or more other biologically active molecules and/or one ormore living cells or tissues.

In other illustrative embodiments, bone resorption is influenced by theadministration of cytokines that increase osteoclast activity including,but not limited to, interleukin-1, M-CSF, tumor nevrosis factor, and/orinterleukin-6. In other embodiments, bone resportion is influenced bythe administration of cytokines that decrease osteoclast activityincluding, but not limited to, interlekin-4, gamma-interferon, and/ortransforming growth factor-beta. In yet other embodiments, boneresorption is influenced by other humoral factors including, but notlimited to, leukotrienes, arachidonic metabolites, and/or prostaglandinsand their inhibitors and including 5-lipoxygenase enzyme inhibitors.

In yet other illustrative embodiments, bone formation is influenced bythe administration of factors that promote osteoblast activity andproliferation including, but not limited to, insulin-like growth factorsI and II, transforming growth factor-beta, acidic and basic fibroblastgrowth factor, platelet-derived growth factor, and/or bone morphogenicproteins.

Bone pore size and porosity influence bone restructuring throughmodifications in bone resorption and/or deposition. Since the size ofthe pores in the bone impacts new bone growth, decreasing the pore sizeand/or the percent of porosity of the bone in the cage reduces orprevents restructuring. In contrast, increasing the pore size and/or thepercent porosity of the bone in the cage enhances restructuring. Thebone cage can be constructed such that the pore size and porosity isapproximately uniform through out the cage, or that the pore size andporosity varies depending on the location. Varying the pore size and/orporosity in discrete locations leads to partial restructuring (eitherpartial enhancement or partial prevention).

In illustrative embodiments, the pore size is approximately 1 nm to 10nm, 1 nm to 20 nm, 1 nm to 25 nm, 1 nm to 50 nm, 1 nm to 100 nm, 1 nm to150 nm, 15 nm to 50 nm, 50 nm to 100 nm, 25 nm to 100 nm, 50 nm to 150nm, or 25 n to 150 nm. In other illustrative embodiments, the pore sizemay be larger, for example approximately 150 nm to 500 nm, 250 nm to 750nm, or 500 nm to 1,500 nm, in one or more locations. This may, forexample, allow partial restructuring in these one or more locations.

In other illustrative embodiments, the pore size may be approximately150 nm to 500 nm, 250 nm to 750 nm, or 500 nm to 1,500 n. In otherillustrative embodiments, the pore size may be smaller, for exampleapproximately 1 nm to 20 nm, 1 nm to 25 nm, 1 nm to 50 nm, 1 nm to 100nm, 1 nm to 150 nm, 15 nm to 50 nm, 50 nm to 100 nm, 25 nm to 100 nm, 50nm to 150 nm, or 25 nm to 150 nm. This may, for example, prevent orreduce restructuring in these one or more locations.

In illustrative embodiments, the porosity is approximately 1% to 15%, 3%to 12%, 5% to 10%, 1% to 3%, 1% to 5%, or 1% to 10% in one or morelocations. In other embodiments, the porosity may be a greaterpercentage in one or more locations, for example approximately 40% to95%, 50% to 90%, 60% to 75%, 15% to 90%, and 25% to 90%. This may, forexample, allow partial restructuring in these one or more locations.

The type of bone used in the fabrication of the cage influences bonerestructuring through modifications in bone resorption and/ordeposition. Measurements of the influence on bone restructuring of eachtype of bone are performed in vitro, as well as in pre-clinical andclinical studies. Different bone types and/or sources have adifferential ability to support restructuring. As a result, bonerestructuring can be partially or completely reduced, or alternatively,partially or completely enhanced depending on the bone chosen. Inaddition, different bone types/sources can be used in discrete locationsin the bone cage to enhance or prevent/decrease bone restructuring.

In illustrative embodiments, studies assessing the ability of new boneor bone cells to restructure a variety of artificial and/or anorganicbone in bone transplant patients or in vitro culture have shown, forexample, that implantation of Bio-Oss lead to limited, reduced or absentrestructuring compared with other artificial or natural organic boneoptions such as Algipore (Clin. Oral Implants Res. (2004) 15:96-100; J.Mater. Sci. Mater. Med. (2005) 16:57-66). Since these studies have alsoidentified artificial bone that encourages restructuring, as doesnatural bone, the bone cage could be designed with portions that areresistant to restructuring as well as portions that encouragerestructuring as desired.

In other illustrative embodiments, bone restructuring is modified bymaking the bone cage from cortical bone, or trabecular or cancellousbone. The choice of bone will impact the extent of restructuring sincecortical bone is generally less porous than trabecular or cancellousbone. In addition, discrete parts of the bone cage could be formed fromone type of bone or another to influence the restructuring of discretelocations.

In yet other embodiments, bone restructuring is modified by the locationof implantation. Bone restructuring is greater when the bone isimplanted in bone rather than other locations. The type of bone the bonecage is implanted in will also influence the extent of restructuring. Inillustrative embodiments, the bone cage is implanted in bone, forexample cortical, or cancellous or trabecular bone. In otherembodiments, the bone cage is implanted in non-bone tissues including,for example, liver, muscle, lung, or fat.

Immunogenicity of the bone cage influences bone restructuring throughmodifications in bone resorption and/or deposition by osteoblasts andosteoclasts, as well as through immune mechanisms. Methods ofinfluencing the immunogenicity of cells are known in the art.Illustrative examples include, but are not limited to, theimmuno-compatibility of donor and recipient, the inherent immunogenicityof the bone material or cells, the presence of immune modulatorycompounds, and genetic modifications.

In some embodiments, the bone cage is partially or completelynon-immunogenic with respect to a subject within whom the device isimplanted, or alternatively, is partially or completely recognized asself. In other embodiments, the bone cage is partially or completelyimmunogenic with respect to a subject within whom the device isimplanted, or alternatively, is partially or completely recognized asnon-self. As used herein, the term “non-immunogenic” means that theimmune response, if any, is not such that immune suppressive drugs wouldbe required following implantation of the bone cage.

In some embodiments, bone cage restructuring and immunogenicity ismodified by the immuno-compatibility of donor and recipient. Inillustrative embodiments, bone cages completely or partially made frombone derived from a donor autologous to the recipient of the bone cage,are non-immunogenic and recognized as self. In some embodiments,previously frozen allogeneic bone, as well as xenogeneic or allogeneicanorganic bone, is considered non-immunogenic.

In illustrative embodiments, bone cages are completely or partially madefrom bone derived from a donor allogeneic to the recipient of the bonecage. In some embodiments, in which the bone is from cadavers, andfrozen, de-mineralized, and/or deorganified, immuno-suppressive therapyis not generally required although some recipients may develop anti-HLAantibodies (The Merck Manual of Diagnosis and Therapy. Sec. 12, Ch.149). In other embodiments, in which the allogeneic bone is not frozen,deorganified or demineralized, for example, an immune response mayresult unless modified by other means, such as immuno-suppressivetherapy.

In other illustrative embodiments, bone cages are completely orpartially made from bone derived from a donor xenogeneic to therecipient of the bone cage. In some embodiments, in which the bone isanorganic bovine bone, for example, immuno-suppressive therapy is notrequired, although some recipients may experience a transient macrophageinfiltrate, but no systemic or local immune response (J. Periodontol.(1994) 65:1008-15). In other embodiments, in which the bone cage is madefrom xenogeneic bone that is not anorganic or pre-frozen, for example,the bone cage is immunogenic and not recognized as self.

In yet other embodiments, the bone cage is partially made fromnon-immunogenic bone, such as but not limited to, autologous bone and/orpre-frozen, de-organified, and/or demineralized allogeneic bone, and/oranorganic xenogeneic bone and partially made from immunogeneic bone,such as but not limited to, allogeneic bone that is not pre-frozen,de-organified, and/or de-mineralized and/or xenogeneic bone that is notanorganic. In some embodiments, the immunogenic bone is placed indiscrete locations to encourage restructuring. In other embodiments, thenon-immunogenic bone is place in discrete locations to prevent or reducerestructuring.

In some embodiments, bone cage restructuring and immunogenicity ismodified by the inherent immunogenicity of the bone material or cells.In some embodiments, bone cages are completely or partially made fromstem cells including, but not limited to mesenchymal, fetal, cord blood,and/or hematopoietic stem cells. In other embodiments, bone cages arecompletely or partially made from differentiated stem cells such as bonecells, including but not limited to, osteoblasts and/or osteoclasts,fibroblasts, or endothelial cells. In some embodiments, the cells areautologous, allogeneic, or xenogeneic as relates to a subject in whom orwhich they are implanted.

In illustrative embodiments, the bone cage is composed of autologous,allogeneic, xenogeneic and/or artificial bone in which autologous,allogeneic, and/or xenogeneic stem cells have been cultured. In someembodiments, the stem cells have been induced to differentiate into, forexample, bone cells including but not limited to osteoblasts and/orosteoclasts. In yet other embodiments, stem cells are cultured indiscrete areas of the bone cage. In some embodiments, the autologous,allogeneic and/or xenogeneic mesenchymal stem cells partially orcompletely decrease the immunogenicity of part, or all, of the bonecage.

Stem cells generally have decreased immunogenicity and can inducetransplant tolerance. For example, hematopoietic stem cells are known toinduce tolerance as can embryonic stem cells (Expert Opin. Biol. Ther.(2003) 3:5-13). In addition, transplanted allogeneic mesenchymal stemcells demonstrate a lack of immune recognition and clearance (Blood(2005) 105:1815-1822; Bone Marrow Transplant (2002) 30:215-222; Proc.Natl. Acad. Sci. USA (2002) 99:8932-8937) as well as being useful ingraft-versus-host disease (Lancet (2004) 363:1439-1441). Mesenchymalstem cells do not activate alloreactive T cells even when differentiatedinto various mesenchymal lineages (Exp. Hematol. (2000) 28:875-884; Exp.Hematol. (2003) 31:890-896), and suppress proliferation of allogeneic Tcells in an MHC-independent manner (Transplantation (2003) 75:389-397;Blood (2005) 105:1815-1822).

In some illustrative embodiments, the bone cage is composed ofautologous, allogeneic, xenogeneic and/or artificial bone in whichautologous, allogeneic, and/or xenogeneic bone cells have been cultured.The bone cells may include, but are not limited to osteoblasts andosteoclasts. In some embodiments, the bone cells are cultured indiscrete areas of the bone cage. In illustrative embodiments, bone cagescreated from autologous, allogeneic, xenogeneic and/or artificial bone,in which allogeneic or xenogeneic (to a subject in which it is to beimplanted) bone cells are propagated, increases the immunogenicity ofthe bone cage when implanted in the subject.

In some embodiments, bone cage restructuring and/or immunogenicity ismodified by the presence of immuno-modulatory compounds. These includeimmuno-suppressive as well as immuno-stimulatory compounds, both ofwhich are known in the art. Immuno-suppressive compounds decreaseimmunogenicity and hence decrease restructuring, whileimmuno-stimulatory compounds increase immunogenicity and hence increaserestructuring. The immuno-modulatory compounds may be administeredsystemically to a subject before, during and/or after implantation ofthe bone cage using methods known in the art. The compounds can beadsorbed onto the surface of the bone cage, placed inside it as one ofthe one or more biologically active molecules, or secreted from the oneor more living cells or tissues. In an embodiment in which the one ormore immuno-modulatory compounds are adsorbed onto the bone cage, theycould be adsorbed to one or more discrete locations on the bone cage.

In illustrative embodiments, the immuno-suppressive compounds include,but are not limited to, corticosteroids, such as prednisolone ormethylprednisolone. In other illustrative embodiments the immunestimulatory and/or inflammatory molecules include, but not limited to,tumor necrosis factor α, interferon γ, interleukin 2, and/or one or moreselections. Other appropriate compounds are known in the art by healthprofessionals and can be found, for example, in the Physician's DeskReference.

In illustrative embodiments, immune stimulatory and/or inflammatorymolecules may be applied to discrete locations on the bone cage. In someembodiments, this results in partial or complete restructuring of thediscrete area. In other illustrative embodiments, immuno-suppressivecompounds may be applied to discrete locations on the bone cage. In someembodiments, this prevents or reduces restructuring of the bone cage inat least those locations.

In some embodiments, the bone cage comprises cells that have beengenetically modified. In some embodiments, the genetically modifiedcells include, but are not limited to, stem cells, bone cells, cellscomprising the semi-permeable component, and/or one or more living cellsor tissues.

In illustrative embodiments, genetic modification of cells influencesbone restructuring and/or immunogenicity. In some embodiments, geneticmodification of cells influences bone resorption and/or deposition. Inother illustrative embodiments, genetic modification of cells stimulatesor inhibits immune reactions. In yet other embodiments, geneticmodification of cells influences the permeability and/or theimmuno-isolatory aspects of the semi-permeable component. In otherembodiments, genetic modification of cells results in the release,secretion, diffusion and/or deposition of one or more biologicallyactive molecules. In yet other embodiments, genetic modification ofcells influences the binding of one or more biologically activemolecules to the bone cage including, but not limited to, the bone walland/or the semi-permeable component.

In some embodiments, the bone cage comprises genetically modified stemcells including, but not limited to, embryonic, fetal, mesenchymal,and/or hematopoietic stem cells. In some embodiments, the stem cells arenon-differentiated. In other embodiments, the stem cells are stimulatedto differentiate. In illustrative embodiments, the stem cells arenon-differentiated mesenchymal stem cells. In other embodiments, themesenchymal stem cells have been differentiated into cells selected fromthe group consisting of osteoblast, osteoclast and endothelial cells.

In some embodiments, cells are genetically modified to increase ordecrease bone restructuring. In other embodiments, stem cells, such asmesenchymal stem cells, are genetically modified to be more or lessosteoconductive when differentiated into osteoblasts or other componentsof bone. Methods for genetic modification of mesenchymal stem cells areknown in the art (Ann. Biomed. Eng. (2004) 32:136-47; Biochem.Biophysica Acta (2005) September 15 Epub; Cloning Stem Cells (2005) &:154-166).

Methods for modifying the osteoconduction of cells are known in the art.For example, bone morphogenetic protein-2 (BMP-2) an osteoinductiveagent, up-regulates the expression of osteogenic phenotypes, and inducesbone nodule formation in a dose-dependent manner (Spine (2004)29:960-5). Ciz, an inhibitor of osteoblast differentiation, interfereswith bone morphogenic protein signaling, which leads to increased bonemass. In illustrative embodiments, a BMP and/or Ciz gene is transducedinto cells and/or its expression up-regulated. Alternatively, a BMPand/or Ciz gene is deleted from the cells by genetic knock out or iRNA,and/or its expression down-regulated by methods known in the art.

In other embodiments, cells are genetically modified to increase ordecrease immunogenicity and/or an immune response. In illustrativeembodiments, cells including, but not limited to stem cells, bone cells,cells of the semi-permeable component, and/or the one or more livingcells or tissues, are genetically modified to express immune recognitionmarkers of the host, to secrete and/or express anti-inflammatorymolecules, and/or to express or secrete immune-stimulatory molecules.

In some embodiments, the bone cage partially or completely surroundsand/or is surrounded by a semi-permeable component. In otherembodiments, the bone cage partially or completely encloses and/or isenclosed by a semi-permeable component. In some embodiments, thesemi-permeable component is partially or completely comprised of thebone wall of the bone cage. In other embodiments, the semi-permeablecomponent is partially or completely external to the bone wall of thebone cage. In other embodiments, the semi-permeable component ispartially or completely internal to the bone wall or the bone cage. Insome embodiments, the semi-permeable component partially or completelyencloses one or more living cells or tissues and/or one or morebiologically active molecules.

As used herein, the term “semi-permeable component” means a selectiveimpediment to the passage of fluids and/or substances in the fluids. Insome embodiments, the semi-permeable component prevents the passage ofmacromolecules and cells, but allows the passage of oxygen and/ornutrients. In some embodiments, the passage of one or more biologicallyactive molecules from the cage and/or products released by the one ormore living cells or tissues in the cage is allowed. In otherembodiments, the passage of macromolecules, or macromolecules and cellsis allowed.

In some embodiments, the semi-permeable component includes, but is notlimited to, the bone wall, one or more semi-permeable membranes, cellswith tight junctions, one or more plasma membranes, one or moreintracellular membranes, one or more red blood cell ghosts, and one ormore aggregated platelets or other cells. In some embodiments, thesemi-permeable component is comprised of cells that are autologous,allogeneic, or xenogeneic with respect to a subject within whom or whichthey may be implanted.

In some embodiments, part, or all, of the semi-permeable component ispartially or completely non-immunogenic and/or is recognized as self bya subject within whom or which it is implanted. In other embodiments,part, or all, of the semi-permeable component is partially or completelyimmunogenic and/or is recognized as non-self by a subject within whom orwhich it is implanted.

In other embodiments, the semi-permeable component is comprised of cellsthat are cultured in vitro. In some embodiments, the semi-permeablecomponent is comprised of cells that are genetically engineered. In someembodiments, some or all of the cells are genetically engineered torelease, secrete, deliver, diffuse, and/or provide one or morebiologically active molecules. In some embodiments, some or all of thecells are genetically engineered to be less immunogenic or to be moreimmunogenic. In yet other embodiments, some or all of the cells aregenetically engineered to increase or decrease bone restructuringincluding, but not limited to, bone deposition and bone resorption. Insome embodiments, the semi-permeable component is designed to at leastpartially or completely enhance restructuring.

In some embodiments, the semi-permeable component is a semi-permeablemembrane. In illustrative embodiments, the semi-permeable membraneincludes, but is not limited to, artificial membranes, biologicalmembranes, and/or a combination of artificial and biologically-derivedcomponents. The manufacture and use of artificial semi-permeablemembranes is known in the art (Cell Transplant (2001) 10:3-24). Knownartificial semi-permeable membranes include, but are not limited to,hydrogel membranes (Biochim. Biophys. Acta (1984) 804:133-136; Science(1991) 254:1782-4; J. Biomed. Mater. Res. (1992) 26:967-977) andultrafiltration membranes (Diabetes (1996) 45:342-347; J. Clin. Invest.(1996) 98:1417-1422; Transplantation (1995) 59:1485-1487; J. Biomech.Eng. (1991) 113:152-170), both which have been employed in theimmuno-isolation of xenografts, for example (Ann. NY Acad. Sci. (1999)875:7-23). The membranes can be made, for example, from polymer filmsand thermoplastic hollow fibers. In addition, biological semi-permeablemembranes are used to encapsulate islet cells followed by implantation(World J. Gastroenterol. (2005) 11:5714-5717).

In other embodiments, the semi-permeable component is partially orcompletely composed of cells with tight junctions. As used herein, theterm “tight junction” or zonula occludens is the intercellular junctionthat regulates diffusion between cells and allows the formation ofbarriers that can separate compartments of different composition. Theintercellular gate formed by tight junctions is size and ion selective,among other things.

In some embodiments, the cells with tight junctions include, but are notlimited to, epithelial and/or endothelial cells, or a combination. Bothepithelial cells and endothelial cells are known to form tight junctionsbetween cells (Methods (2003) 30:228-234).

In yet other embodiments, the semi-permeable component is comprised ofcells with tight junctions where the cells are stem cells, or aredifferentiated from stem cells. In illustrative embodiments, stem cellsare cultured in vitro to confluency on the interior and/or exterior of abone scaffold of the desired shape and composition. In some embodiments,the stem cells include, but are not limited to, one or more ofmesenchymal, embryonic, fetal, or hematopoietic stem cells. In someembodiments, the stem cells are stimulated to differentiate. In someembodiments, the stem cells differentiate into one or more ofendothelial cells and epithelial cells. In some embodiments, the stemcells differentiate into bone cells, including but not limited to,osteoblasts or osteoclasts. In other embodiments the stem cells do notdifferentiate into bone cells.

Methods for differentiating mesenchymal stem cells into endothelialcells (Basic & Clin. Pharmacol. & Toxicol. (2004) 95:209-214) andhematopoietic stem cells into epithelial stem cells are known in theart. Stem cells are known to be relatively non-immunostimulatory, and toretain this characteristic following differentiation.

In yet other embodiments, the semi-permeable component is a plasmamembrane. In some embodiments, the plasma membrane is made from red cellghosts. Red cell ghosts are created by removal of the erythrocytecytoplasm by lysis followed by size-exclusion chromatography. In someembodiments, one or more red cell ghosts encapsulate the one or morebiologically active molecules and/or the one or more living cells and/ortissues. Methods of using red cell ghosts for drug delivery are known inthe art (Expert Opinion on Drug Delivery (2005) 2:311-322; Drug Delivery(2003) Taylor & Francis eds. 10(4):277-282; BioDrugs (2004) 18:189-198).

In other embodiments, the one or more red cells ghosts are fused to forman internal or external continuous or semi-continuous membrane. In someembodiments, the fused red blood cell ghosts encapsulate the one or morebiologically active molecules and/or the one or more living cells and/ortissues.

In other embodiments, the semi-permeable component is an aggregate ofplatelets. In an illustrative embodiment, the bone cage is coatedinternally and/or externally with a platelet aggregating compound onwhich platelets aggregate in vitro and/or in vivo. In some embodimentsthe platelet aggregating compound includes, but is not limited to,fibrin, fibrinogen and/or thrombin. For example, fibrinogen is known toplay a role in platelet aggregation (Coll. Anthropol. (2005) 29:341-9).

In yet other embodiments, the semi-permeable component is comprised ofendothelial cells having one or more characteristics of a blood brainbarrier. In the body, the blood brain barrier is a specialized system ofcapillary endothelial cells that protect the brain from harmfulsubstances in the blood stream, while supplying the brain with therequired nutrients for proper function. The blood brain barrier strictlylimits transport into the brain through both physical (tight junctions)and metabolic (enzymes) barriers (Ann. Rev. Pharmacol. Toxicol. (2003)43:629-56).

In some embodiments, the semi-permeable component is comprised ofendothelial cells having one or more characteristics of cerebralcapillary endothelial cells. In some embodiments, the semi-permeablecomponent includes one or more characteristics of cerebral capillaryendothelium that is different from the characteristics of peripheralcapillary endothelium or non-cerebral capillary epithelium. In otherembodiments, the semi-permeable membrane includes one or morecharacteristics of cerebral capillary endothelium that is not acharacteristic of peripheral capillary endothelium or non-cerebralcapillary epithelium.

The microvasculature of the central nervous system differs fromperipheral tissue endothelium in several ways, including in tightjunctions, cytoplasm, mitochondria, enzymatic barriers, wall thickness,and polarity.

In some embodiments, the semi-permeable component comprises endothelialcells with interendothelial tight junctions. Cerebral capillaryendothelial cells contain tight junctions, which seal cell-to-cellcontacts between adjacent endothelial cells forming a continuous bloodvessel. The tight junctions between blood brain barrier endothelialcells lead to high endothelial electrical resistance, in the range of1500 to 2000 Ω-cm² (pial vessels) and as high as 8000 Ωcm² (non-pialvessels), as compared with 3-33 Ωcm² in other tissues (J. Gen. Physiol.(1981) 77:349-71; J. Neurochem. (1986) 46:1732-1742; J. Physiol. (1990)429:47-62). The elevated resistance in brain endothelium leads to lowparacellular (between cell) permeability.

In some embodiments, the interendothelial tight junctions have anendothelial resistance greater than 33 Ωcm², greater than 50 Ωcm²,greater than 100 Ωcm², greater than 200 Ωcm², greater than 500 Ωcm²,greater than 750 Ωcm², or greater than 1000 Ωcm². In other embodiments,the interendothelial tight junctions have an endothelial resistancebetween 33 Ωcm² and 8000 Ωcm², 50 Ωcm² and 8000 Ωcm², 100 cm² and 8000Ωcm², 200 Ωcm² and 8000 Ωcm², 500 Ωcm² and 8000 Ωcm², 750 ωcm² and 8000Ωcm², or 1000 Ωcm² and 8000 Ωcm². In yet other embodiments, theinterendothelial tight junctions have an endothelial resistance between1500 to 2000 Ω-cm², 1000 to 2500 Ω-cm², 500 to 2500 Ω-cm², 100 to2000-cm², 1500 to 5000 Ω-cm², or 1500 to 8000 Ω-cm².

In some embodiments, the interendothelial tight junctions include one ormore integral membrane proteins different from or not present inperipheral capillary endothelium or non-cerebral capillary endothelium.The intracellular clefts of cerebral capillary endothelium are around200 angstroms wide, and are constricted by macula and zonula adhaerensand zonula occludens (Circ. Res. (1976) 38:404-411). The adhaerensjunction is around 200 angstroms, while the occludens junction (tightjunction) is essentially completely occluded. Tight junction membranecomponents include, but are not limited to, occludin, claudins(multi-gene family of at least 20 isomers, including claudin-1 andclaudin-5), zonula occludens (at least ZO-1/2/3), junctional adhesionmolecules (JAM), and microdomains on the cell membrane rich incholesterol, which contain caveolin (J. Cell Biol. (1999) 147:891-903;Trends Cell Biol. (1999) 9:268-273; J. Cell Biol. (1998) 141:199-208; J.Cell Sci. (2000) 113:1771-1781).

In some embodiments, the semi-permeable component includes endothelialcells with cytoplasm that is different from the cytoplasm of peripheralcapillary endothelium or non-cerebral capillary endothelium. In someembodiments, the cytoplasm is of uniform thickness. In otherembodiments, the cytoplasm contains very few pinocytic vesicles. In yetother embodiments, the cytoplasm lacks fenestrations. The cytoplasm ofthe endothelial cells of the blood brain barrier is of uniform thicknesswith very few pinocytic vesicles (hollowed out portion of cells membranefilled with fluid, forming a vacuole which facilitates nutrienttransport), and lacks fenestrations (openings). The cytoplasm may haveone seventh of the amount of pinocytic vesicles compared with musclecapillary endothelial cells. Therefore transport across the blood brainbarrier involves translocation through capillary endothelium, theinternal cytoplasmic domain, and then through the ablumenal membrane.

In some embodiments, where the cytoplasm has fewer pinocytic vesiclesthan cytoplasm of peripheral endothelium, there are 10%, 20%, 30%, 50%,75%, 90% or 95% fewer pinocytic vesicles. In other embodiments, thecytoplasm has 10% to 50%, 25% to 50%, 25% to 75%, 50% to 75%, or 75% to95% fewer pinocytic vesicles. In yet other embodiments, the cytoplasmhas one half, one third, one quarter, one fifth, one sixth, one seventh,one eighth, one ninth, or one tenth the number of pinocytic vesiclesfound in muscle capillary endothelium.

In some embodiments, where the cytoplasm has fewer fenestrations thanthe cytoplasm of peripheral endothelium, there are 10%, 20%, 30%, 50%,75%, 80%, 85% 90%, 95%, or 99% fewer fenestrations. In otherembodiments, the cytoplasm has 10% to 50%, 25% to 50%, 25% to 75%, 50%to 75%, 75% to 90%, 85% to 95%, or 95% to 99% fewer fenestrations. Inembodiments where the cytoplasm lacks fenestrations, the presence offenestrations is undetectable by methods known in the art, or there is adecrease in fenestrations that is 95% to 99% or greater compared withperipheral endothelium.

In some embodiments, the semi-permeable component has endothelial cellscontaining a greater number and/or volume of mitochondria as comparedwith peripheral endothelium or non-cerebral capillary endothelium. Theincrease in mitochondria and therefore energy potential, may be requiredto allow active transport of nutrients to the brain from the blood(Proc. Soc. Exp. Biol. Med. (1975) 149:736-738; Ann. Neurol. (1977)1:409-417). In some embodiments, there are 5 to 6 times moremitochondria per capillary cross section than in peripheral capillaryendothelium. In other embodiments, there are approximately 2, 3, 4, 5,6, 7, 8, 9, or times more mitochondria per capillary cross section thanin peripheral capillary endothelium. In yet other embodiments, there are2 to 4, 3 to 5, 4 to 7, 8 to 10, 3 to 8, or 2 to 6 times moremitochondria per capillary cross section than in peripheral capillaryendothelium.

In some embodiments, the semi-permeable component includes endothelialcells with elevated metabolizing enzymes. The cerebral endotheliacontain an enzymatic barrier capable of metabolizing drug and nutrients,including but not limited to, neuroactive solutes (Brain Res. Brain Res.Rev. (1991) 16:65-82; J. Neurochem. (1993) 60:793-803; J. Pharmacol.Exp. Ther. (1994) 270:675-680). Enzymes in elevated concentrationscompared with non-neuronal capillary endothelium include, but are notlimited to, γ-glutamyl transpeptidase (γ-GTP), alkaline phosphatase, andaromatic acid decarboxylase. Other specific enzymes expressed by brainendothelial cells include, but are not limited to, monoamine oxidases,epoxy hydrolase, and endopeptidases. One or more of the enzymes may beabsent or at lower levels in non-neuronal capillary endothelium. In someembodiments, the metabolizing enzymes are present at levels at least10%, 20%, 30%, 50%, 75%, 90%, 100%, 200%, or 500% higher. In otherembodiments, the metabolizing enzymes are present at levels 10% to 50%,25% to 50%, 25% to 75%, 50% to 75%, 75% to 100%, 100% to 200%, or 200%to 500% higher.

In some embodiments, the semi-permeable component has endothelium with adecreased capillary wall thickness. Cerebral capillary endothelial cellsmay have an approximately 39% decrease in the wall thickness comparedwith muscle capillary endothelial cells (Microvasc. Res. (1985)30:99-115). In some embodiments, the wall thickness is decreased by 10%,20%, 30%, 40%, 50%, 60%, or 75% compared with muscle capillaryendothelial cells. In other embodiments, the wall thickness is decreasedby 10% to 50%, 25% to 50%, 25% to 75%, 50% to 75%, or 30% to 50%.

In some embodiments, the semi-permeable component has endothelium with adefined polarity reflecting the polarity of the lumenal and ablumenalsurfaces of the blood brain barrier. As used herein, the term “lumenal”refers to the internal surface of the capillary endothelium. The lumenalsurface of the endothelium forms a hollow tube through which blood flowsin vivo. As used herein, the term “ablumenal” refers to the exteriorsurface of the capillary endothelium. The ablumenal surface of theendothelium is in contact with structures of the brain in vivo,including astrocytes, pericytes, and neurons.

In some embodiments, polarity of the cerebral capillary endothelium andthe semi-permeable membrane is defined through differences in one ormore enzymes, receptors, or transporters present on one surface ascompared with the other surface. In other embodiments, polarity isdefined through presence or absence of one or more enzymes, receptors,or transporters on one surface as compared with the other surface. Forexample, γ-glutamyl transpeptidase and alkaline phosphatase are usuallyfound associated with the lumenal portion of the blood brain barrier,while Na⁺—K⁺-ATPase and the sodium dependent neutral amino acidtransporter are usually associated with the ablumenal membrane (BrainRes. (1980) 192:17-28). The glucose receptor (GLUT-1) typically has a3:1 ratio of distribution, ablumenal to lumenal surface (Proc. Natl.Acad. Sci. USA (1991) 88:5779-5783). The P-glycoprotein (P-gp) drugefflux transporter exists primarily at the lumenal membrane surface.

In some embodiments, the lumenal surface is oriented toward the internalcavity of the semi-permeable membrane of the device. In otherembodiments the ablumenal surface is oriented toward the internal cavityof the semi-permeable membrane of the device.

In some embodiments, the semi-permeable component has endothelium withone or more transport systems that are different from those innon-neural capillary endothelium. In some embodiments, one or more ofthe transport systems are not present in non-neural capillaryendothelium. In the blood brain barrier, and in contrast to peripheralendothelium, the rate of pinocytosis is minimal, and free membranediffusion applies mainly to small lipophilic molecules, including butnot limited to, ethanol and nicotine. Active or catalyzed transportsystems include carrier-mediated bidirectional transport, typicallyunidirectional efflux transport, and receptor-mediated transport byendocytosis and transcytosis (Cellular and Molecular Neurobiology (2005)25:59-127). Carrier-mediated bidirectional transport (responsible fornutrient uptake in the brain) includes, but is not limited to, glucosetransporter (GLUT-1), monocarboxylic acid transporter (MCT1), largeneutral amino acid transporter (LAT1), and sodium-coupled nucleosidetransporter (CNT2). Efflux transport typically removes metabolites andxenobiotics from the brain, and/or prevents metabolites and xenobioticsfrom crossing the endothelium to reach the brain, and includes, but isnot limited to, P-glycoprotein and MRP-1 multidrug resistance proteins,brain multidrug resistance protein (ABCG2/BCRP), and organicanion-transporting polypeptide (OATP2). Receptor-mediated transport byendocytosis and transcytosis supplies the brain with peptides andproteins including, but not limited to, low-density lipoproteins,transferrin, leptin, and insulin.

Illustrative embodiments showing blood brain barrier devices are shownin FIG. 5. FIG. 5A shows a cross-section of one orientation of an ovoidblood brain barrier endothelium device 501 comprised of endothelialcells 510 optionally containing interendothelial tight junctions 520, Pglycoprotein efflux pump 530 at interior plasma membrane surfaces 590,ion transporters 540 and glucose receptor 550 at exterior plasmamembrane surfaces 595, pinocytic vesicles 560, mitochondria 570, andnuclei 580. FIG. 5B shows a cross-section of another orientation of anovoid blood brain barrier endothelium device 502 comprised ofendothelial cells 510 optionally containing interendothelial tightjunctions 520, the P glycoprotein efflux pump 530 at exterior plasmamembrane surfaces 595, ion transporters 540 and glucose receptor 550 atinterior plasma membrane surfaces 590, pinocytic vesicles 560,mitochondria 570, and nuclei 580.

In some embodiments, the endothelium that makes part or all of thesemi-permeable component is autologous, allogeneic, or xenogeneic withrespect to a subject. The endothelium may be cultured in vitro. Theendothelial cells may be from primary brain endothelium, from peripheralendothelium, from an endothelial cell line, or from stem cells. Theendothelium may be genetically engineered, designed to partially orcompletely prevent restructuring, designed to partially or completelyenhance restructuring, be immunogenic, be non-immunogenic, and/or berecognized as self.

In some embodiments, the blood brain barrier device may be implantableand biocompatible, and may also include a structural component. Theendothelium may at least partially or completely form an internalcavity. The structural component may at least partially or completelyform an internal cavity. The combination of the endothelium and thestructural component may partially or completely form an internalcavity. In some embodiments, the structural component may surround theendothelium. In other embodiments, the endothelium may surround thestructural component. The structural component may be at least partiallycomposed of bone or of cartilage.

In some aspects, a method of making a blood brain barrier deviceincludes, but is not limited to, forming a semi-permeable component fromendothelial cells, configuring the semi-permeable component to at leastpartially form an internal cavity, and engineering the endothelial cellsto have one or more characteristics of a blood brain barrier membrane.In some embodiments, at least one of the one or more characteristics ofthe blood brain barrier membrane is not a characteristic of peripheralendothelium or non-cerebral capillary endothelium. In some embodiments,a method of making a device includes culturing cells in vitro. In someembodiments, the cells include, but are not limited to, endothelialcells, stem cells, and brain endothelial cells. In yet otherembodiments, a method of making a device includes differentiatingendothelial cells from stem cells, and/or culturing stem cells in vitro.In some embodiments, the blood brain barrier device includes cells thatare isolated from autologous, allogeneic, or xenogeneic donors.

In yet other embodiments, a method of making a device includesconfiguring the semi-permeable component to at least partially form aninternal cavity by culturing endothelial cells on a scaffold to at leastpartially form the internal cavity. In some embodiments, a method ofmaking a device includes configuring the scaffold to at least partiallyform the internal cavity.

In yet other embodiments, a method of making a device includes selectinga structural component, and combining the structural component and thesemi-permeable component. In some embodiments, a method of making adevice includes selecting a structural component, and placing thesemi-permeable component in the structural component. In someembodiments, a method of making a device includes selecting a structuralcomponent, and placing the structural component in the semi-permeablecomponent. In yet other embodiments, a method of making a deviceincludes selecting a structural component, and at least partiallysurrounding the structural component with the semi-permeable component.In yet other embodiments, a method of making a device includes selectinga structural component, and at least partially surrounding thesemi-permeable component with the structural component.

In yet other embodiments, a method of making a device includesidentifying one or more biologically active molecules, and providing theone or more biologically active molecules to the internal cavity of thedevice. In yet other embodiments, a method of making a device includesidentifying one or more living cells or tissues, and providing the oneor more living cells or tissues to the internal cavity. In someembodiments, the one or more living cells and tissues produce the one ormore biologically active molecules.

In other aspects, a method for delivering one or more biologicallyactive molecules to a subject includes, but is not limited to,identifying one or more biologically active molecules useful to asubject, and implanting in the subject one or more of the blood brainbarrier devices described in this disclosure. In some embodiments, amethod of making a device includes adding the one or more biologicallyactive molecules to the one or more devices before or after implantationof the device in a subject in need of the biologically active molecules.In some embodiments, a method of making a device includes identifyingone or more living cells or tissues, and providing the one or moreliving cells or tissues to the one or more devices. In some embodiments,the one or more living cells and tissues produce the one or morebiologically active molecules.

In yet other aspects, a method of assembling a device for delivering oneor more biologically active molecules to a subject, includes identifyingthe one or more biologically active molecules, selecting one or more ofthe blood brain barrier devices described herein, and providing the oneor more biologically active molecules to the device. In someembodiments, a method of making a device includes identifying one ormore one or more living cells or tissues, and providing the one or moreliving cells or tissues to the one or more devices. In some embodiments,the one or more living cells and tissues produce the one or morebiologically active molecules.

Models for the blood-brain barrier have been developed in vitro (ATLA(2004) 32:37-50; NeuroAIDS (1998) 1(4): 1-6; Proc. Natl. Acad. Sci. USA(1998) 95:1840-1845; Cellular and Molecular Neurobiology (2005)25:59-127; Brain Research Reviews (2005) 50:258-265; Journal ofPharmacology and Experimental Therapeutics (2006) 316:79-86; Cellularand Molecular Neurobiology (2005) 25:201-210; Biol. Pharm. Bull. (2001)24:111-118). The characteristics studied include, but are not restrictedto, restrictive paracellular permeability, physiologically realisticarchitecture, expression of the functional transporter mechanismspresent in vivo, transendothelial electrical resistance (TEER),endothelial permeability coefficient (P_(e)), and ease of culture. Thein vitro blood brain barrier models can derive from isolated braincells, primary (subpassaged) culture cells, immortalized brainendothelial cells, cell lines of non-cerebral origin, co/multi cultures,and cells free systems, including partition coefficients and artificialmembranes.

In yet other embodiments, the semi-permeable component comprisesepithelial cells having one or more characteristics of the bloodcerebrospinal fluid barrier. In the body, the blood cerebrospinal fluidbarrier is a specialized system including modified choroidal epithelialcells. The choroid plexus forms the cerebrospinal fluid and activelyregulates the concentration of molecules in the cerebrospinal fluid. Thecells of the choroid plexus are modified and have epithelialcharacteristics. The cells have microvilli on the cerebrospinal fluidside, basolateral interdigitations, and abundant mitochondria. Thechoroidal epithelial cells form tight junctions preventing mostmacromolecules from effectively passing into the cerebrospinal fluidfrom the blood.

In some embodiments, the semi-permeable component comprises epithelialcells having one or more characteristics of choroid plexus epithelium.In some embodiments, the semi-permeable component includes one or morecharacteristics of choroid plexus epithelium that is different from thecharacteristics of peripheral epithelium or non-choroid plexusepithelium. In other embodiments, the semi-permeable component includesone or more characteristics of the choroid plexus epithelium that is nota characteristic of peripheral epithelium, and/or non-neural epithelium,and/or non-choroidal plexus epithelium. In some embodiments, thesemi-permeable membrane at least partially, or completely, forms aninternal cavity.

Characteristics of the choroid plexus epithelium that may be differentfrom the peripheral epithelium include one or more of interepithelialtight junctions, interepithelial electrical resistance, apicalmicrovilli, basolateral interdigitations, mitochondria, active orcatalyzed transport systems, and polarity between apical and basalsurfaces.

In some embodiments, the semi-permeable component comprises epithelialcells having interepithelial tight junctions. The choroidal plexusepithelium contains tight junctions (spot welds) at the apical zone ofneighboring epithelial cells (Pharmaceutical Research (2005)22:1011-1037). The tight junctions at least partially occlude or blockthe passage of water soluble agents (depending on molecular size) fromone side of the barrier to the other. There is paracellular diffusion ofsmall hydrophilic solutes through choroidal plexus tight junctions. Insome embodiments, the interepithelial tight junctions include one ormore integral membrane proteins not present in peripheral epithelium ornon-choroidal plexus epithelium. The one or more integral membraneproteins may include, but are not limited to, one or more of occludin,claudin-1, and zona occludens-1 (ZO-1).

In some embodiments, the interendothelial tight junctions lead to anincreased epithelial electrical resistance compared with peripheralepithelium, or non-choroidal plexus epithelium. In some embodiments, theinterepithelial tight junctions have an epithelial resistance greaterthan 33 Ωcm², greater than 50 Ωcm², greater than 100 Ωcm², greater than200 Ωcm², greater than 500 Ωcm², greater than 700 Ωcm², greater than 800Ωcm², or greater than 1000 Ωcm². In other embodiments, theinterepithelial tight junctions have an endothelial resistance between33 Ωcm² and 3000 Ωcm², 50 Ωcm² and 2500 Ωcm², 100 Ωcm² and 2000 Ωcm²,200 Ωcm² and 2000 Ωcm², 50 Ωcm² and 250 Ωcm², 150 Ωcm² and 250 Ωcm², 700Ωcm² and 800 Ωcm², or 1000 Ωcm² and 2000 Ωcm². In yet other embodiments,the interepithelial tight junctions have an epithelial resistancebetween 1500 to 2000 Ωcm², 1000 to 2500 Ωcm², 500 to 2500 Ωcm², 50 to2000 Ωcm², 100 to 2000 Ωcm², 800 to 2000 Ωcm² or 1500 to 5000 Ωcm².

In some embodiments, the semi-permeable component comprises epithelialcells with active or catalyzed transport systems. The epithelial cellsof the choroid plexus secrete cerebrospinal fluid by a process thatinvolves the transport of Na⁺, Cl⁻ and HCO₃ ⁻ from the blood to theventricles of the brain (Neuroscience (2004) 129:957-970). Theunidirectional transport of ions is achieved due to the polarity of theepithelium. The ion transport proteins in the blood-facing (basolateral)membrane are different from those in the ventricular (apical) membrane.The apical membrane of the choroid plexus has ion transportersincluding, but not limited to, Na⁺—K⁺ ATPase, K⁺ channels, andNa⁺-2Cl—K⁺ cotransporters, and aquaporin 1 to mediate water transport.The basolateral membrane of the choroid plexus has ion transportersincluding, but not limited to, Cl⁻—HCO₃ exchangers, a variety of Na⁺coupled HCO₃ ⁻ transporters, and K⁺—Cl cotransporters.

In some embodiments, the semi-permeable membrane is composed ofepithelial cells with one or more characteristics of the choroidalepithelium, including differential localization of ion transportproteins between interior and exterior membranes. In some embodiments,the ion channels of the apical membrane of the choroidal plexus arepartially or completely oriented toward the internal cavity of thedevice. In other embodiments, the ion channels of the apical membrane ofthe choroidal plexus are partially or completely oriented toward theexterior of the device. In some embodiments, the ion channels of thebasolateral membrane of the choroidal plexus are partially or completelyoriented toward the internal cavity of the device. In other embodiments,the ion channels of the basolateral membrane of the choroidal plexus arepartially or completely oriented toward the exterior of the device. Insome embodiments, the localization and/or action of ion transportproteins may result in partially or completely unidirectional transportof ions. The unidirectional transport may occur in either orientation,partially or completely toward the internal cavity, or partially orcompletely toward the exterior of the device.

In yet other embodiments, the semi-permeable component is composed ofepithelial cells with one or more characteristics of the choroidalepithelium, including nutrient transporters. In the brain, the choroidplexus epithelium facilitates nutrient transport from the blood(basolateral surface) to the ventricle (cerebrospinal fluid surface)with transporters that include, but are not limited to, ascorbic acidspecific transporter (SVCT2) and the myo-inosital-Na⁺-cotransporter(SMIT; Advanced Drug Delivery Reviews (2004) 56:1859-1873). In someembodiments, the nutrient transporters include, but not limited to, theascorbic acid specific transporter and themyo-inosital-Na⁺-cotransporter. In some embodiments, the nutrienttransporters of the apical membrane of the choroidal plexus arepartially or completely oriented toward the internal cavity of thedevice. In other embodiments, the nutrient transporters of the apicalmembrane of the choroidal plexus are partially or completely orientedtoward the exterior of the device. In some embodiments, the nutrienttransporters of the basolateral membrane of the choroidal plexus arepartially or completely oriented toward the internal cavity of thedevice. In other embodiments, the nutrient transporters of thebasolateral membrane of the choroidal plexus are partially or completelyoriented toward the exterior of the device. In some embodiments, thelocalization and/or action of nutrient transporters may result inpartially or completely unidirectional transport of nutrients. Theunidirectional transport may occur in either orientation, partially orcompletely toward the internal cavity, or partially or completely towardthe exterior of the device.

In some embodiments, the semi-permeable component is composed ofepithelial cells with one or more characteristics of the choroidalepithelium, including organic transport proteins. In the brain, thegrowing families of organic anion transporting polypeptides (Oatp/OATP)and multidrug resistance-associated proteins (Mrp/MRP) mediate the fluxof amphiphilic, anionic and in some cases even cationic molecules acrossthe blood cerebrospinal fluid barrier (Advanced Drug Delivery Reviews(2004) 56:1859-1873). These transport proteins also include, but are notlimited to, the MDR1 (multidrug resistance) P glycoprotein (Pgp), andthe multidrug resistance-associated protein (MRP), a homologousATP-binding cassette transporter (Proc. Natl. Acad. Sci. (1999)96:3900-3905). The transporters are involved in the clearance ofpotentially toxic compounds, as well as many drugs (including, but notlimited to, β-lactam antibiotics, cephalosporins, cytostatic drugs, andantiviral agents), from the cerebrospinal fluid. In some embodiments,the organic transport proteins include, but are not limited to, organicanion transporting polypeptides, multidrug resistance-associatedproteins, ATP-binding cassette transporters, and multidrug resistanceproteins. In some embodiments, the organic transport proteins of theapical membrane of the choroidal plexus are partially or completelyoriented toward the internal cavity of the device, In other embodiments,the organic transport proteins of the apical membrane of the choroidalplexus are partially or completely oriented toward the exterior of thedevice. In some embodiments, the organic transport proteins of thebasolateral membrane of the choroidal plexus are partially or completelyoriented toward the internal cavity of the device. In other embodiments,the organic transport proteins of the basolateral membrane of thechoroidal plexus are partially or completely oriented toward theexterior of the device. In some embodiments, the localization and/oraction of organic transport proteins may result in partially orcompletely unidirectional transport of toxic compounds. Theunidirectional transport may occur in either orientation, partially orcompletely toward the internal cavity, or partially or completely towardthe exterior of the device.

In yet other embodiments, the semi-permeable membrane is composed ofepithelial cells' with one or more characteristics of the choroidalepithelium, including differential localization of organic transportproteins between interior and exterior membranes of the device. Thedifferential localization of transport proteins in the choroid plexusepithelium has a different polarity than that observed in the epitheliumof the kidneys or liver (Microscopy Research and Technique (2001)52:60-64). The Na⁺/K⁺-ATPase is localized at the apical plasma membranedomain, and the first step in organic anion removal from thecerebrospinal fluid requires substrate uptake across the apical plasmamembrane domain of choroid plexus epithelial cells. Therefore, unlike inthe kidneys, the “kidney-like” excretory system for organic anions islocated at the apical side of the choroid plexus epithelium.

In some embodiments, the semi-permeable membrane has epithelial cellscontaining members of the organic anion transporter (OAT) family thatare oriented toward the internal cavity. In other embodiments, thesemi-permeable membrane has epithelial cells containing members of theorganic anion transporter (OAT) family that are oriented away from theinternal cavity. In other embodiments, the semi-permeable membrane hasepithelial cells containing members of the organic cation transporter(OCT) family that are oriented toward the internal cavity. In yet otherembodiments, the semi-permeable membrane has epithelial cells containingmembers of the organic cation transporter (OCT) family that are orientedaway from the internal cavity.

In some embodiments, the choroid plexus epithelial cells that form thesemi-permeable component may contain liver-like transport properties. Inanother embodiment, one or more liver-like organic anion transportersare expressed in the choroidal epithelium forming the semi-permeablemembrane of the device. These transporters may include, but are notlimited to, the family of organic anion transporting proteins (Oatps;OATPs). The organic anion transporting proteins may be differentiallyexpressed (have a polar distribution) between the apical and basolateralsurfaces of the choroid plexus epithelium. For example, organic aniontransporting protein 1 may be expressed at the apical, and organic aniontransporting protein 2 at the basolateral surface.

In some embodiments, the semi-permeable membrane has epithelial cellscontaining members of the organic anion transporting protein family thatare oriented toward or away from the internal cavity. In otherembodiments, the semi-permeable membrane has epithelial cells containingmembers of the organic anion transporting protein family that have apolar orientation, with one or more family members oriented toward theinternal cavity, and a different one or more family members orientedaway from the internal cavity. In some embodiments, the localizationand/or action of organic anion transporting protein family may result inpartially or completely unidirectional transport of toxic compounds. Theunidirectional transport may occur in either orientation, partially orcompletely toward the internal cavity, or partially or completely towardthe exterior of the device.

In yet other embodiments, the choroid plexus epithelial cells that formthe semi-permeable component may contain one or more members of theATP-binding cassette transporter family. In some embodiments, the one ormore members of the ATP-binding cassette transporter family may includeone or more members of the multidrug resistance protein (Mrp/MRP)subfamily. At least one member of the multidrug resistance proteinsubfamily (MRP1) has been localized to the basolateral membrane of thechoroid plexus epithelium. MRP1 is a versatile multispecific organicsubstrate pump that can mediate ATP-dependent efflux of a large varietyof organic compounds, and in the presence of normal intracellularglutathione, many neutral and basic organic compounds. In someembodiments, the semi-permeable membrane has epithelial cells containingmembers of the ATP-binding cassette transporter family and/or one ormore members of the multidrug resistance protein (Mrp/MRP) subfamilythat are oriented toward or away from the internal cavity.

In yet other embodiments, the choroid plexus epithelial cells that formthe semi-permeable component may contain one or more members of themultidrug resistance (MDR) P glycoprotein family. In brain choroidplexus epithelium, the MDR1 P glycoprotein has been localized to theapical plasma membrane and a subapical vesicular compartment (Proc.Natl. Acad. Sci. (1999) 96:3900-3905). This localization would confer anapical to basal transepithelial permeation barrier for MDR1 substrates.In some embodiments, the semi-permeable membrane has epithelial cellscontaining members of the multidrug resistance P glycoprotein familythat are oriented toward or away from the internal cavity.

In some embodiments, the active or catalyzed transport systems of theepithelial cells that form the semi-permeable component of the deviceinclude one or more of organic anion and cation transporters, ABCtransporters, ATA2, PepT2, transthyretin, transferrin receptor, and Na+,K+-ATPase.

In some embodiments, the semi-permeable component has epithelial cellsthat produce and secrete cerebrospinal fluid proteins including, but notlimited to, transthyretin (pre-albumin), ascorbic acid, andmyo-inositol.

In some embodiments, the semi-permeable component has infoldings orinterdigitations. In some embodiments, the infoldings orinterdigitations are oriented toward the internal cavity. In otherembodiments, the infoldings or interdigitations are oriented toward theexterior of the device, or away from the internal cavity. In someembodiments, the infoldings or interdigitations form the internalcavity. In other embodiments, the infoldings or interdigitations formthe exterior of the device. In vivo, the choroid epithelium containsextensive basolateral infoldings that provide a substantial surface fortransport (Pharmaceutical Research (2005) 22:1011-1037). In someembodiments, there are at least approximately 2, 5, 10, 25, 50 or 100times more interdigitations per epithelial cross section than innon-choroidal epithelium. In yet other embodiments, there are 2 to 5, 5to 10, 10 to 50, 50 to 100, or 100 to 1000 times more interdigitationsper epithelial cross section than in non-choroidal epithelium.

In other embodiments, the semi-permeable component includes epithelialcells with microvilli and/or with greater numbers and/or volume ofmicrovilli as compared with non-choroidal epithelium. In the brain, thechoroid epithelium contains lush apical membrane microvilli that providea massive surface area for molecular fluxes (Pharmaceutical Research(2005) 22:1011-1037). In some embodiments, the microvilli are partiallyor completely oriented toward the internal cavity. In other embodiments,the microvilli are partially or completely oriented toward the exteriorof the device, or away from the internal cavity. In some embodiments,the microvilli partially or completely form the internal cavity. Inother embodiments, the microvilli partially or completely form theexterior of the device. In some embodiments, there are at leastapproximately 2, 5, 10, 25, 50 or 100 times more microvilli perepithelial cross section than in non-choroidal epithelium. In yet otherembodiments, there are 2 to 5, 5 to 10, 10 to 50, 50 to 100, or 100 to1000 times more microvilli per epithelial cross section than innon-choroidal epithelium.

In yet other embodiments, the semi-permeable component has epithelialcells containing a greater number and/or volume of mitochondria ascompared with peripheral epithelium. The increase in mitochondria, andtherefore energy potential, provide the energy for substantial secretionand reabsorption (Pharmaceutical Research (2005) 22:1011-1037). In someembodiments, there are approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 timesmore mitochondria per epithelial cross section than in non-choroidalepithelium. In yet other embodiments, there are 2 to 4, 3 to 5, 4 to 7,8 to 10, 3 to 8, or 2 to 6 times more mitochondria per epithelial crosssection than in non-choroidal epithelium.

In some embodiments, the semi-permeable component has epithelium with adefined polarity reflecting the polarity of the apical and basolateralsurfaces of the choroid plexus epithelium. As used herein, the term“basolateral” refers to the internal surface of the choroid plexusepithelium that faces the capillary endothelium. The basolateral surfaceof the epithelium surrounds the fenestrated capillary through whichblood flows in vivo. As used herein, the term “apical” refers to theexterior surface of the choroid plexus epithelium. The apical surface ofthe epithelium is in contact with the cerebrospinal fluid that bathesthe brain, including the ventricles.

In some embodiments, polarity of the choroid plexus epithelium and thesemi-permeable component is defined through differences in membranestructure and/or location of tight junctions on or near one surface ascompared with another surface. In other embodiments, polarity is definedthrough presence or absence of membrane structures and/or location oftight junctions on one surface as compared with the other surface. Forexample, microvilli are usually associated with the apical surface ofthe choroid plexus epithelium, and basolateral interdigitations areassociated with the basal surface of the choroid plexus epithelium.Tight junctions are typically formed between cells on adjacent surfacestoward the apical surface of the choroid plexus epithelium.

In some embodiments, polarity of the choroid plexus epithelium and thesemi-permeable component is defined through differences in one or moretransporter systems, which may include but are not limited to, iontransport proteins, nutrient transporters, organic transport proteinsand ATP-binding cassette members, present on one surface as comparedwith the other surface. In other embodiments, polarity is definedthrough presence or absence of one or more transporter systems which mayinclude but are not limited to, ion transport proteins, nutrienttransporters, organic transport proteins and ATP-binding cassettemembers, on one surface as compared with the other surface. For example,ion transporters including, but not limited to, Na⁺—K⁺ ATPase, K⁺channels, and Na⁺-2Cl—K⁺ cotransporters, and aquaporin 1 are typicallylocated on the apical membrane of the choroid plexus epithelium. Iontransporters including, but not limited to, Cl⁻-HCO₃ exchangers, avariety of Na⁺ coupled HCO₃ ⁻ transporters, and K⁺—Cl cotransporters aretypically located on the basolateral membrane of the choroid plexusepithelium. Organic transport proteins including, but not limited to,the organic anion transporter are typically located on the apicalmembrane of the choroid plexus epithelium. Organic anion transportersincluding, but not limited to, organic anion transporting proteins havemembers of the family preferentially localized to either the apical orbasal membranes. ATP-binding cassette members including, but not limitedto, multidrug resistance protein 1 are typically located on thebasolateral surface of the choroid epithelium, while P glycoprotein istypically loicated on the apical surface of the choroid plexusepithelium.

In yet other embodiments, polarity of the choroid plexus epithelium andthe semi-permeable component is defined through differences in secretionand/or presence or absence of secretion of proteins from one surface ascompared with the other. For example, cerebrospinal fluid,transthyretin, ascorbic acid, and myo-inositol are typically secretedfrom the apical surface.

In some embodiments, the basolateral surface is oriented toward theinternal cavity of the semi-permeable component of the device. In otherembodiments the apical surface is oriented toward the internal cavity ofthe semi-permeable component of the device.

Illustrative embodiments showing blood cerebrospinal fluid barrierdevices are shown in FIG. 6. FIG. 6A shows a cross-section of oneorientation of an ovoid choroid plexus epithelium device 601 comprisedof epithelial cells 610 optionally containing exterior plasma membranemicrovilli 620, interior plasma membrane interdigitations 630,interepithelial tight junctions 640 near the external surface, iontransport systems 650 on interior and exterior plasma membranes, organictransport systems on interior and exterior plasma membranes 660,mitochondria 670 and nuclei 680. FIG. 6B shows a cross-section ofanother orientation of an ovoid choroid plexus epithelium device 602comprised of epithelial cells 610 optionally containing interior plasmamembrane microvilli 620, exterior plasma membrane interdigitations 630,interepithelial tight junctions 640 near the internal surface, iontransport systems 650 on interior and exterior plasma membranes, organictransport systems 660 on interior and exterior plasma membranes,mitochondria 670 and nuclei 680.

In some embodiments, the epithelium that makes part or all of thesemi-permeable component is autologous, allogeneic, or xenogeneic withrespect to a subject. The epithelium may be cultured in vitro. Theepithelial cells may be from primary brain epithelium, from peripheralepithelium, from an epithelial cell line, or from stem cells. Theepithelium may be genetically engineered, designed to partially orcompletely prevent restructuring, designed to partially or completelyenhance restructuring, be immunogenic, be non-immunogenic, and/or berecognized as self.

In some embodiments, the blood cerebrospinal fluid barrier device may beimplantable and biocompatible, and may also include a structuralcomponent. The epithelium may at least partially or completely form aninternal cavity. The structural component may at least partially orcompletely form an internal cavity. The combination of the epitheliumand the structural component may partially or completely form aninternal cavity. In some embodiments, the structural component maysurround the epithelium. In other embodiments, the epithelium maysurround the structural component. The structural component may be atleast partially composed of bone or of cartilage.

In some aspects, a method of making a blood cerebrospinal fluid barrierdevice includes, but is not limited to, forming a semi-permeablemembrane from epithelial cells, configuring the semi-permeable membraneto at least partially form an internal cavity, and engineering theepithelial cells to have one or more characteristics of a bloodcerebrospinal fluid barrier membrane. In some embodiments, at least oneof the one or more characteristics of the blood cerebrospinal fluidbarrier membrane is not a characteristic of peripheral epithelium. Insome embodiments, a method of making a device includes culturing cellsin vitro. In some embodiments, the cells include, but are not limitedto, epithelial cells, stem cells, and brain epithelial cells. In yetother embodiments, a method of making a device includes differentiatingepithelial cells from stem cells, and/or culturing stem cells in vitro.In some embodiments, the blood cerebrospinal fluid barrier deviceincludes cells that are isolated from autologous, allogeneic, orxenogeneic donors.

In yet other embodiments, a method of making a device includesconfiguring the semi-permeable membrane to at least partially form aninternal cavity by culturing epithelial cells on a scaffold to at leastpartially form the internal cavity. In some embodiments, a method ofmaking a device includes configuring the scaffold to at least partiallyform the internal cavity.

In yet other embodiments, a method of making a device includes selectinga structural component, and combining the structural component and thesemi-permeable membrane. In some embodiments, a method of making adevice includes selecting a structural component, and placing thesemi-permeable membrane in the structural component. In someembodiments, a method of making a device includes selecting a structuralcomponent, and placing the structural component in the semi-permeablemembrane. In yet other embodiments, a method of making a device includesselecting a structural component, and at least partially surrounding thestructural component with the semi-permeable membrane. In yet otherembodiments, a method of making a device includes selecting a structuralcomponent, and at least partially surrounding the semi-permeablemembrane with the structural component.

In yet other embodiments, a method of making a device includesidentifying one or more biologically active molecules, and providing theone or more biologically active molecules to the internal cavity of thedevice. In yet other embodiments, a method of making a device includesidentifying one or more living cells or tissues, and providing the oneor more living cells or tissues to the internal cavity. In someembodiments, the one or more living cells and tissues produce the one ormore biologically active molecules.

In other aspects, a method for delivering one or more biologicallyactive molecules to a subject includes, but is not limited to,identifying one or more biologically active molecules useful to asubject, and implanting in the subject one or more of the bloodcerebrospinal fluid barrier devices described in this disclosure. Insome embodiments, a method of making a device includes adding the one ormore biologically active molecules to the one or more devices before orafter implantation of the devices in a subject in need of thebiologically active molecules. In some embodiments, a method of making adevice includes identifying one or more living cells or tissues, andproviding the one or more living cells or tissues to the one or moredevices. In some embodiments, the one or more living cells and tissuesproduce the one or more biologically active molecules.

In yet other aspects, a method of assembling a device for delivering oneor more biologically active molecules to a subject, includes identifyingthe one or more biologically active molecules, selecting one or more ofthe blood cerebrospinal fluid barrier devices described herein, andproviding the one or more biologically active molecules to the device.In some embodiments, a method of making a device includes identifyingone or more one or more living cells or tissues, and providing the oneor more living cells or tissues to the one or more devices. In someembodiments, the one or more living cells and tissues produce the one ormore biologically active molecules.

Models for the blood cerebrospinal fluid barrier using, among others,isolated choroid plexus, cultured epithelial cells from the choroidplexus, and immortalized choroid plexus epithelial cell lines, have beendeveloped (Advanced Drug Delivery Reviews (2004) 56:1875-1885; AdvancedDrug Delivery Reviews (2004) 56:1859-1873; Proc. Natl. Acad. Sci. (1999)96:3900-3905).

In other embodiments, the bone cage comprises one or more biologicallyactive molecules. In some embodiments, the one or more biologicallyactive molecules are surrounded by the semi-permeable component. Inother embodiments, the one or more biologically active molecules arebound to the bone cage. In other embodiments, the bone binds one or morebiologically active molecules. In some embodiments, the bone binds thesemolecules following their release from the bone cage and/or living cellsand/or tissues. In some embodiments, the one or more biologically activemolecules comprise part of the bone wall. In other embodiments, the oneor more biologically active molecules are bound to the semi-permeablecomponent and/or one or more living cells or tissues. In yet otherembodiments, the one or more biologically active molecules are releasedfrom, provided by, secreted from, and/or diffuse from cells of the bonewall, the semi-permeable component, and/or one or more living cells ortissues.

As used herein, the term “biologically active molecules” includes anymolecule that has a measurable biological action in a subject. Forexample, biologically active molecules would include, but not be limitedto, any molecules described in this disclosure including, but notlimited to, molecules that enhance or reduce bone restructuringincluding bone resorption and deposition, and/or that enhance or reducean immune response. In illustrative embodiments, these biologicallyactive molecules would include, but not be limited to, pharmaceuticallyacceptable compounds including parenteral drugs, nutrients, and vitaminsincluding, but not limited to those described in this disclosure for thetreatment of particular diseases or disorders.

In illustrative embodiments, the one or more biologically activemolecules include, but are not limited to, hormones such as adrenalin,adrenocorticotropic hormone (ACTH), aldosterone, antidiuretic hormone(Vasopressin), calcitonin, cholecystokinin, cortisol, insulin, gastrin,glucagon, glucocorticoids, gonadotropin-releasing hormone, luteinizingand follicle stimulating hormones, growth hormone, estrogen,testosterone and thyroid hormone. In other embodiments, the one or morebiologically active molecules include, but are not limited to, hormonesof the gut, such as gastrin, secretin, cholecystokinin, somatostatin andneuropeptide Y. In other embodiments, the one or more biologicallyactive molecules include, but are not limited to hormones of thehypothalamus such as thyrotropin-releasing hormone (TRH),gonadotropin-releasing hormone (GnRH), growth hormone-releasing hormone(GHRH), ghrelin, corticotropin-releasing hormone (CRH), somatostatin,dopamine, antidiuretic hormone (ADH), obestatin and oxytocin. In otherembodiments, the one or more biologically active molecules include, butare not limited to hormones of the kidney such as renin, erythropoietin(EPO) and calcitriol. In other embodiments, the one or more biologicallyactive molecules include, but are not limited to hormones of the liversuch as insulin-like growth factor-1 (IGF-1), angiotensinogen, andthrombopoietin. In other embodiments, the one or more biologicallyactive molecules include, but are not limited to hormones of thepituitary including those from the anterior lobe such as thyroidstimulating hormone (TSH), follicle-stimulating hormone (FSH),luteinizing hormone (LH), prolactin (PRL), growth hormone (GH), andadrenocorticotropic hormone (ACTH), as well as the posterior lobe suchas antidiuretic hormone (ADH) and oxytocin. In other embodiments, theone or more biologically active molecules include, but are not limitedto, hormones of the reproductive system such as estrogens, progesterone,testosterone, and anabolic steroids. In other embodiments, the one ormore biologically active molecules include, but are not limited to,leptin, ghrelin, obestatin, resistin, melanocyte-stimulating hormone(MSH), parathyroid hormone, melatonin and prolactin.

In other embodiments, the bone cage comprises one or more living cellsor tissues. In some embodiments, a semi-permeable component surroundsthe one or more living cells or tissues. In some embodiments, the cellsare autologous, allogeneic, or xenogeneic with respect to a subjectwithin whom or which they may be implanted. In some embodiments, thecells are cultured in vitro. In some embodiments the cells arenon-immunogenic and/or are recognized as self by a subject within whomor which they is implanted. In some embodiments, the one or more livingcells or tissues have been genetically engineered. In some embodiments,the one or more living cells or tissues have been genetically engineeredto release, provide, diffuse and/or extrude the one or more biologicallyactive molecules.

In some embodiments, the one or more living cells and/or tissuesinclude, but are not limited to, cells and/or tissues that produce,express and/or secrete immune/inflammation-related, biochemicalfunction-related, metabolism-related, and/or hormone-relatedbiologically active molecules. In illustrative embodiments, the one ormore living cells and/or tissues include, but are not limited to,bacteria, yeast, islet cells, liver cells, thyroid cells, bone cells,and/or neural cells.

Other aspects include methods for delivering one or more biologicallyactive molecules to a subject. The one or more biologically activemolecules to be delivered to the subject are identified and/or selectedby methods well-known in the art, for example by health care workersincluding, but not limited to, physicians responsible for the health ofthe subject. One or more of the bone cages described above are selectedfor delivery of the one or more biologically active molecules. The oneor more biologically active molecules may be provided with or added tothe bone cages, and/or released from one or more living cells or tissuesprovided with or added to the bone cages, and/or released from the cellscomprising the semi-permeable component provided with or added to thebone cages. The one or more bone cages containing the one or morebiologically active molecules and/or living cells or tissues and/orsemi-permeable component are implanted in the subject to allow deliveryof the one or more biologically active molecules.

Yet other aspects include methods for assembling a device for deliveringone or more biologically active molecules to a subject. The one or morebiologically active molecules to be delivered to the subject areidentified and/or selected by methods well-known in the art, for exampleby health care workers including, but not limited to, physiciansresponsible for the health of the subject. One or more of the bone cagesdescribed above are selected for delivery of the one or morebiologically active molecules. The one or more biologically activemolecules may be provided with or added to the bone cages, and/orreleased from one or more living cells or tissues provided with or addedto the bone cages, and/or released from the cells comprising thesemi-permeable component provided with or added to the bone cages. Theone or more bone cages containing the one or more biologically activemolecules and/or living cells or tissues and/or semi-permeable componentare implanted in the subject to allow delivery of the one or morebiologically active molecules.

Other aspects include methods of using one or more bone cages to treat,ameliorate, and/or prevent one or more diseases and/or disorders. Insome embodiments, the one or more diseases and/or disorders include, butare not limited to, immune-related, biochemical function-related,metabolism-related, hormone-related, wound healing, burns, surgicalincisions, joint ailments, bone-related, obesity, addiction, and/orneurological-related.

In illustrative embodiments, use of bone cages in the treatment,amelioration and/or prevention of immune and/or inflammation-relateddiseases and/or disorders includes, but is not limited to, enhancing theimmune response to treat for example malignancies and/or infections, andcreation of tolerance to treat, for example, allergies, asthma, andautoimmune disorders.

In illustrative embodiments, use of bone cages in the treatment,amelioration and/or prevention of biochemical function-related and/ormetabolism-related diseases and disorders includes, but is not limitedto aspects of liver and/or pancreas dysfunction. In illustrativeembodiments for liver dysfunction, allogeneic or xenogeneic liver cells,optionally including stem cells, are placed within one or more bonecages to perform toxin processing, metabolize protein, metabolizecarbohydrates, and/or treat lysosomal storage disorders and fatty acidoxidation defects. In illustrative embodiments for pancreas dysfunction,allogeneic or xenogeneic Islet cells are placed within one or more bonecages to produce insulin.

In illustrative embodiments, use of bone cages in the treatment,amelioration and/or prevention of hormone-related diseases and disordersincludes, but is not limited to, hypothyroidism, panhypopituitarism,osteoporosis, adrenal insufficiency, and/or sex hormone deficiency. Insome embodiments, allogeneic and/or xenogeneic donor cells replace thedeficient hormones. In other embodiments, genetically engineered cells,for example stem cells, bacteria and/or yeast, replace the deficienthormones.

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, and 4H show tables 401, 402, 403, 404,405, 406, and 407, respectively, that describe diseases and disorders ina column entitled Disease 410 that can be treated, ameliorated and/orprevented using one or more of the bone cages described in thisdisclosure. For example, cells or tissues containing non-defectiveversions of the system or enzyme described in the column entitledDefective Enzyme or System 420 can be administered to a subject in needof such treatment by implantation of one or more bone cages. Subjects inneed of treatment are identified according to their symptoms, forexample, as described in the column entitled Symptoms 430. In addition,a current treatment, shown in the column entitled Treatment 440, can beadministered to a subject in need of such treatment by use of one ormore bone cages.

All references are hereby incorporated by reference herein in theirentirety.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

What is claimed is:
 1. A method for delivering one or more biologicallyactive molecules to a subject, comprising: providing one or morebiologically active molecules; providing one or more devices with asemi-permeable component including a confluent tissue coating of bloodbrain barrier capillary endothelial cells; at least one structuralcomponent that is at least partially composed of porous bone that is incontact with and supports the semi-permeable component; thesemi-permeable component substantially surrounds the at least onestructural component; and an internal cavity that is substantiallysurrounded by the semi-permeable component and at least a portion of theporous bone; the one or more biologically active molecules included inthe pores and/or the cavity of the one or more devices; and implantingin the subject one or more of the devices containing the biologicallyactive molecules; wherein at least one characteristic of the blood brainbarrier capillary endothelial cells is different from the at least onecorresponding characteristic of peripheral capillary endothelial cells.2. The method of claim 1, further comprising: providing the one or morebiologically active molecules to the internal cavity.
 3. The method ofclaim 2, wherein providing the one or more biologically active moleculesto the internal cavity comprises: providing the one or more biologicallyactive molecules to the internal cavity through one or more closableopenings; and closing the one or more closable openings.
 4. The methodof claim 2, wherein providing the one or more biologically activemolecules to the internal cavity comprises: providing one or more livingcells or tissues to the internal cavity, wherein the one or more livingcells or tissues release the one or more biologically active molecules.5. the method of claim 4, wherein the one or more living cells ortissues are engineered to release the one or more biologically activemolecules.
 6. The method of claim 1, wherein the semi-permeablecomponent substantially surrounds an interior of the internal cavity ofthe one or more devices.
 7. The method of claim 1, wherein thesemi-permeable component substantially surrounds an exterior of the oneor more devices including the internal cavity.
 8. A method fordelivering one or more biologically active molecules to a subject,comprising: providing one or more devices with a semi-permeablecomponent including: a confluent tissue coating of a blood brain barriercapillary endothelial cells; at least one structural component that isat least partially composed of porous bone that is in contact with andsupports the semi-permeable component; the semi-permeable componentsubstantially surrounds the at least one structural component; whereinthe one or more devices form an internal cavity that is substantiallysurrounded by the semi-permeable component and at least a portion of theporous bone: implanting in the subject one or more of the devices;providing the one or more biologically active molecules into theinternal cavity following implantation of the one or more devices intothe subject; wherein at least one characteristic of the blood brainbarrier capillary endothelial cells is different from the at least onecorresponding characteristic of peripheral capillary endothelial cells.9. A method of assembling a device for delivering one or morebiologically active molecules to a subject, comprising: providing one ormore biologically active molecules; and providing one or more deviceswith a semi-permeable component including a confluent tissue coating ofblood brain barrier capillary endothelial cells; at least one structuralcomponent that is at least partially composed of porous bone that is incontact with and supports the semi-permeable component; thesemi-permeable component substantially surrounds the at least onestructural component; an internal cavity that is substantiallysurrounded by the semi-permeable component and at least a portion of theporous bone; and the one or more biologically active molecules includedin the pores and/or the cavity of the one or more devices; wherein atleast one characteristic of the one or more characteristics of the bloodbrain barrier capillary endothelial cells is different from at least onecorresponding characteristic of peripheral capillary endothelial cells.10. The method of claim 9, further comprising: providing the one or morebiologically active molecules to the internal cavity.
 11. The method ofclaim 10, further comprising: identifying one or more living cells ortissues to release the one or more biologically active molecules; andproviding the one or more living cells or tissues to the internalcavity.
 12. The method of claim 11, wherein the one or more living cellsor tissues are engineered to release the one or more biologically activemolecules.
 13. The method of claim 10, wherein providing the one or morebiologically active molecules to the internal cavity comprises:providing one or more living cells or tissues to the internal cavity,wherein the one or more living cells or tissues release the one or morebiologically active molecules.
 14. The method of claim 9, wherein theone or more devices include one or more closable openings.
 15. Themethod of claim 14, further comprising: closing the one or more closableopenings.